Synthetic peptides specific for the detection of antibodies to HCV

ABSTRACT

The present invention relates to a method for the detection in body fluids of antibodies to hepatitis C virus (HCV), also known as a non-A non-B hepatitis (NANBH) virus and to the diagnosis of NANBH by the use of a composition of synthetic peptides. Each of these peptides has an amino acid sequence corresponding to immunodominant regions of a fusion protein and a non-structural polypeptide of HCV, SOD/HCV C100 and a postulated HCV structural (core) protein. More specifically, the present invention is directed to the use of a group of synthetic peptides in a prescribed sequence or their analogues for the detection of antibodies to HCV in body fluids. The detection method includes an enzyme-linked immunosorbent assay (ELISA), and other forms of immunoassay procedures. 
     The present invention also relates to a method for generating high titer antibodies to HCV in healthy mammals, including humans, by the use of compositions containing these synthetic peptides, analogues or mixtures thereof, in a free, conjugated or polymeric form as key components in synthetic vaccines for the prevention of non-A non-B hepatitis (NANBH).

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation in part application of copending application Ser.No. 07/481,348 and now abandoned, filed Feb. 16, 1990, and applicationSer. No. 07/510,153 and now abandoned, filed Apr. 16, 1990.

INTRODUCTION

The present invention relates to peptide compositions specific for thediagnosis and prevention of hepatitis C virus (HCV) infection, or non-Anon-B hepatitis (NANBH). More particularly, the present invention isdirected to synthetic peptide compositions which are specific for thedetection of antibodies to HCV in body fluids and immunoassays using thesame. The invention also includes the use of the synthetic peptidecompositions as antigens for eliciting the production of monoclonal andpolyclonal antibodies against HCV and as immunogens in vaccines for theprevention of NANBH or HCV infection.

In the 1940's, two independent investigators concluded that there wereat least two types of viral hepatitis, designated as A and B (HAV andHBV) and that infection by one type, either HAV or HBV, did not conferthe patient with cross-immunity (1-3). It was only in the 1970's withthe introduction of serologic markers for hepatitis A and hepatitis Bthat it became possible to identify diseases caused by the two virusesand to distinguish between these two types of hepatitis clinically andserologically.

Subsequently, in 1974, Prince et al. suggested that many cases oftransfusion hepatitis could not be attributed to HAV or HBV and werecaused by an agent other than these viruses. They proposed naming theagent hepatitis C virus (HCV) (4). The presence of another hepatitiscausing agent was subsequently confirmed by Alter et al., who reportedthat although the exclusion of commercial blood donors found to carryhepatitis B surface antigen (HBsAg) significantly reduced the frequencyof post-transfusion hepatitis (5), 7 to 10 percent of the 5 millionAmericans who received transfusions each year still developed hepatitis.In 90% of these post-transfusion hepatitis cases, a specific virus,unrelated to HAV, HBV, Epstein-Barr virus, cytomegalovirus or otherviruses which occasionally produce liver diseases, was implicated as theetiologic agent (5). This infection was designated as non-A non-Bhepatitis (NANBH).

Over the years, NANBH has been reported in patients undergoinghemodialysis, recipients of renal transplants (6), intravenous drugabusers (7) and patients in institutions for the mentally retarded (8).Further, nurses caring for patients with NANBH have also been found tocontract this disease.

Epidemiologic evidence suggests that there may be three types of NANBH:the water borne epidemic type; the blood or needle associated type; andthe sporadically occurring community acquired type. However, the numberand precise nature of the causative agents of NANBH still remain notentirely clear.

The acute phase of NANBH is less severe than that of hepatitis B, andthe disease is rarely fatal. However, more than a third of theindividuals who contract NANBH develop a chronic form of the disease inwhich they may remain infectious indefinitely. This chronic state maylead to cirrhosis of the liver and eventually to liver cancer.

Many methods have been developed in an attempt to detect the putativeNANBH viral antigens and antibodies. These include agar-gel diffusion,counter immunoelectrophoresis, immunofluorescence microscopy,immunoelectron microscopy, radioimmunoassay, and enzyme-linkedimmunosorbent assay using crude biologic lysates and antibodies frompatients. However, none of these assays are sufficiently sensitive,specific, and reproducible for use as a diagnostic test for NANBH. Someof the reactivities detected were later attributable to the presence ofantibodies to host cytoplasmic antigens or low levels of arheumatoid-factor-like substance frequently present in patients with orwithout hepatic diseases.

In the absence of a definitive test for NANBH, the diagnosis in the pasthas been one of exclusion. It was based on the clinical presence ofacute hepatitis and the persistent absence of serologic markers forhepatitis A and B, Epstein-Barr virus or cytomegalovirus.

Because no specific test for the detection of antibodies to NANBH or HCVhas been available, the use of nonspecific tests to screen donors hasbeen adopted in the past decade as a means of preventing at least somepost-transfusion NANBH.

One such surrogate test measures liver enzyme levels. The concentrationsof some of the liver enzymes, in particular alanine aminotransferase(ALT), are frequently elevated in the blood of patients with activehepatitis. Two independent studies have shown a correlation betweendonor ALT levels and the incidence of NANBH in transfusion recipients(9-11). However, some studies showed that only about 20 percent of blooddonors who transmitted NANBH have elevated liver enzyme concentrations.Other investigators, furthermore, have found that the liver enzymelevels can be increased by extraneous factors, such as heavy drinking.

Epidemiologic circumstances predisposing donor populations to infectionwith hepatitis B virus may also favor exposure to NANBH agents. A studyconducted by Stevens et al. (12) evaluated the risk factors in donorsfor the presence of antibodies to hepatitis B virus. The resultsindicated that units of blood which were positive for antibodies to thehepatitis B core antigen (anti-HBc) appeared to present a two tothree-fold greater risk of NANBH in the recipients than units withoutanti-HBc. They concluded that anti-HBc screening of donors might preventabout one third of the cases of NANBH attributable to transfusion,whereas ALT screening might prevent nearly one half of the cases of posttransfusion NANBH.

Even with the use of these surrogate tests to establish the diagnosis ofNANBH by exclusion, the correct identification of the NANBHV carrierswas still far from satisfactory. Firstly, there are a significant numberof patients who received blood lacking the surrogate markers and yetdeveloped NANBH. Secondly, there is a minimal overlap between donorswith elevated ALT levels and those with anti-HBc. Lastly, there arerecipients of blood units which were positive for a surrogate marker,but who did not become infected with NANBHV, also known as HCV (13-15).

Thus, there is an urgent demand for a sensitive and specific method toidentify carriers of NANBHV and to screen out contaminated blood orblood products. In addition, there is also a need for an effectivevaccine and/or therapeutic agent for the prevention and/or treatment ofthe disease.

Recently, a group of scientists at Chiron Corp. constructed arandom-primed complementary DNA (cDNA) library from plasma containingthe uncharacterized NANBH agent (16). They screened the library withserum from a patient diagnosed with NANBH and isolated a cDNA clone thatencodes an antigen associated specifically with NANBH. This clone wasfound to be derived from the genome of an agent similar to thetogaviridae or flaviviridae (16). The newly identified NANBH agent wascalled hepatitis C virus (HCV). A specific assay for this blood-borneNANBH virus was developed based on a fusion polypeptide of humansuperoxide dismutase (SOD) and 363 HCV amino acids, designated asSOD/HCV C100-3 (17). SOD/HVC C-100-3 was cultured from a clone ofrecombinant yeast, purified, and used to capture circulating viralantibodies (17). A family of cDNA sequences derived from this hepatitisC virus was subsequently reported in detail (18).

However, the neucleotide sequence of HCV disclosed by the Chiron groupcovers only about 75% of the HCV genome and represents only thenonstructural genes.

More recently Mayumi, et al. determined the 5'-terminal sequence of thegenome of HCV for two distinct HCV strains in human and chimpanzeecarriers (27). The 5'-terminal sequence contained a 5' non-coding regionof at least 324 nucleotides, well preserved in the two strains. Thenon-coding region was followed by a coding region of 1348 nucleotidescontinuing beyond the reported sequence of the prototype HCV whichspanned 7310 nucleotides (18). Based on these results (18,27), HCV isconsidered to possess an uninterrupted open reading frame encoding atleast 2886 amino acid residues.

A comparison of the complete nucleotide sequence of the Hepatitis Cvirus to that of other Flaviviruses (28) has led us to postulate thattwo structural genes encoding for the core (or nucleocapsid protein) andthe envelope proteins were contained in the HCV genome located in theupstream and downstream region respectively of the 5'-terminal sequenceas reported by the Mayumi group (27). By careful analysis of the wholeHCV genome structure and the predicted amino acid sequence encoded inthe structural and non-structural proteins, we have now identified andcharacterized by an extensive series of experiments and throughserological validation, the immunodominant regions of the HCV proteins.

The predicted amino acid sequence of the HCV genome is presented inTable 6, wherein the sequence for (a) is the sequence for J-1 (27, 29),(b) is the sequence for J-4 (27) and (c) is the sequence for theprototype PT (18). These show where conservative substitutions,deletions or substitutions can be made.

                                      TABLE 6                                     __________________________________________________________________________    (a)                                                                              MSTIPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATR                                                                           50                          (b)                                                                              ---N----------------------------------------------                            KTSERSQPRGRRQPIPKVRRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSP                                                                           100                            ------- ---W------A------A---------------L---------                           RGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARA                                                                           150                            --------------------------------------- -----------                           LAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASAYQVRNSTGL                                                                           200                            ------------------------------------I-----E---VS-I                            YHVTNDCPNSSIVYEAHDAILHTPGCVPCVREGNVSRCWVAMTP TVATRD                                                                          250                            -------S--------A-M-M-----------D-S------L---L-A-N                            GKLPATQLRRHIDLLVGSATLCSALYVGDLCGSVFLIGQLFTFSPRRHWT                                                                           300                            ASV-T-TI---V-----A-AF---M-----------V S----------E-                           TQGCNCSIYPGHITGHRMAWDMMMNWSPTAALVMAQLLRIPQAILDMIAG                                                                           350                            V-D---------LS---------------T---VS--------VV--V--                            AHWGVLAGIAYFSMVGNWAKVLVVLLLFAGVDAETIVSGGQAARAMSGLV                                                                           400                            --------L --Y----------I-A----------YT---A-SHTT-T-A                           SLFTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWLAGLIYQHKFNSS                                                                           450                            ---S---S-R---V---------R------D--H--F--A-F-T-R--- -                        (a)                                                                              GCPERLASCRRLTDFDQGWGPISHANGSGPDQRPYCWHYPPKPCGIVPAK                                                                           500                         (b)                                                                              -----M-----IDW-A------TYTEPDS----------A-R-------S                         (c)                                                                              -----------------------Y--------------- -----------                           SVCGPVYCFTPSP                                  550                            Q------------                                                                 -------------VVVGTTDRSGAPTYSWGENDTDVFVLNNTRPPLGNWF                            GCTWMNSTGFTKVCGAPPCVIGGAGNNTLHCPTDCFRKHPDATYSRCGSG                                                                           600                            PWITPRCLVDYPYRLWHWPCTINYTIFKIRMYVGGVEHRLEAACNWTRGE                                                                           650                            RCDLEDR DRSELSPLLLTTTQWQVLPCSFTTLPALSTGLIHLHQNIVDVQ                                                                          700                            YLYGVGSSIASWAIKWEYVVLLFLLLADARVCSCLWMMLLISQAEAALQN                                                                           750                            LVILNAASLAGTHGLVSFLVFFCFAWYLKGKWVPGAVYTFYGMWPLLLLL                                                                           800                            LALPQRAYALDTEVAASCGGVVLVGLMALTLSPYYKRYISWCLWWLQYFL                                                                           850                            TRVQAQLHVWIPPLNVRGGRDAVILLMLAVHPTLVFDITKLLLAV FGPLW                                                                          900                            ILQASLLKVPWFVRVQGLLRFCALARKMIGGHYVQMVIIKLGALTGTYVY                                                                           950                            NHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGL                                                                           1000                           PVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGR                                                                           1050                           DKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQM                                                                           1100                           YTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVR RRGASRG                                                                          1150                           SLLSPROISYLKGSSGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVEN                                                                           1200                           LETTMRSPVFTDNSSPPVVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYK                                                                           1250                           VLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFL                                                                           1300                           ADGGCSGGAYDIIICDELHSTDATSILGIGTVLDQAETAGARLVVLATAT                                                                           1350                           PPGSVTVPHPNIEEVAL STTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKC                                                                          1400                        (a)                                                                              -                                              1450                        (c)                                                                              DELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGYTGDFDS                            ------------------------T-Y-----------------RR----                                                                           1500                           VIDCNTCVTQTVDFSLDPTFTIETITLPQDA VSRTQRRGRTGRGKPGIYR                           --T------A-------------------------S------L-------                                                                           1550                           FVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPV                            --------S--------------- ----A-D-F--------------K--                                                                          1600                           CNDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAP                            ----------------------------------V-----I--    1650                           PPSWDQMWKCLIRLKPTLHGPTPLLYRL GAVQNEITLTHPVTKYIMTCMS                           ADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRVVLSGKPAIIPDREV                                                                           1700                           LYREFDEMEECSQHLPYIENGMMLAENFKQKALGLLQTASRQAEVIAPAV                                                                           1750                           QTNWQKLETFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSP                                                                           1800                           LTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLID                                                                           1850                           ILAGWGAGVAGALVAFKIMSGEVP STEDLVNLLPAILSPGALVVGVVCAA                                                                          1900                           ILRRHVGPGEGAVNWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSS                                                                           1950                           LTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLM                                                                           2000                           PQLPGIPFVSCQRGYKGVWRVDGIMHTRCHCGAEITGHVKNGTMRIVGPR                                                                           2050                           TCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFH                                                                           2100                           YVTGMTTDNLKCPCQV PSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVG                                                                          2150                           LHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVAS                                                                           2200                           SSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKV                                                                           2250                           VILDSFDPLVA EEDEREISVPAEILRKSRRFAQALPVWARPDYNPPLVET                                                                          2300                           WKKPDYEPPVVHGCPLPPPKSPPVPPPRKKR T VVLTESTLSTALAELATR                                                                         2350                           SFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGE PGDPDL                                                                          2400                           SDGSWSTVSSEANAEDVVCCSMSYSWTGALVTPCAA EEQKLPINALSNSL                                                                          2450                           LRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKA                                                                           2500                           NLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVTHINSVWKDLLEDN                                                                           2550                           VT PIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVT                                                                          2600                           KLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTE                                                                           2650                           SDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCR                                                                           2700                           ASRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGDDLVVICESA                                                                           2750                           GVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSC SSNVSVAHDGA                                                                          2800                           GKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMY                                                                           2850                           HFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRL        2889                        __________________________________________________________________________

Synthetic peptides have been increasingly used to map antigenic orimmunogenic sites on the surface of proteins, an approach recentlytermed "site-directed-serology". The present inventor (Wang, C. Y.) anda colleague have taken this approach to identify and characterize highlyantigenic epitopes on the envelope proteins of HIV and to developsensitive and specific immunoassays for the detection of antibodies toHIV (previously designated HTLV-III) (19-21). See also U.S. Pat. No.4,735,896, issued Apr. 5, 1988 and U.S. Pat. No. 4,879,212 issued Nov.7, 1989, the contents of which are, hereby, fully incorporated byreference (22, 23). Subsequently, a series of finely mappedell-characterized HTLV-I/II related synthetic peptides were employed inthe development of synthetic peptide-based diagnostic assays for thedetection of HTLV-1/II antibodies in infected individuals (24, 25). Seealso U.S. Pat. No. 4,833,071 issued May 23, 1989, U.S. Ser. No.07/297,635 filed Jan. 13, 1989 and U.S. Ser. No. 07/469,294 filed Jan.24, 1990. These assays have provided superior sensitivity, excellentspecificity, and, in certain cases, an unmatched capability todifferentiate infections with two closely related viruses, thusovercoming many of the existing problems associated withbiologically-derived tests based on either viral lysate or recombinantDNA-derived protein.

It is, therefore, an objective of the present invention to develop adetection or diagnostic procedure to identify and monitor HCV infectionearly in the disease cycle.

Another objective is to develop a test procedure that is highlysensitive and accurate.

A further objective is to chemically synthesize a test reagent which canthen be used to detect the presence of antibodies to HCV in body fluidsand diagnose NANBH.

Another objective is to develop a vaccine which, when introduced intohealthy mammals, including humans, will stimulate production ofefficacious antibodies to HCV, thereby providing protection against HCVinfection.

A further objective is to provide a synthetic immunogen which can beused in mammals for the development of monoclonal and polyclonalantibodies to HCV.

LIST OF REFERENCES

1. MacCallum F O, Bauer D J: Homologous serum jaundice: transmissionexperiments with human volunteers. Lancet. 1:6222 (1944).

2. Havens W P: Experiment in cross immunity between infectious hepatitisand homologous serum jaundice. Proc Soc Exp Biol Med. 59:148 (1945).

3. Krugman S, Giles J P, Hammond J: Infectious hepatitis. Evidence fortwo distinctive clinical, epidemiological and immunological types ofinfection. JAMA, 200:365 (1967).

4. Prince A M, Brotman B, Grady G F, et al: Long-incubationpost-transfusion hepatitis without serological evidence of exposure tohepatitis-B virus. Lancet, 2:241 (1974).

5. Alter H J, Purcell R H, Holland P V, et al: Clinical and serologicalanalysis of transfusion-associated hepatitis. Lancet, 2:838 (1975).

6. Galbraith R M, Protmann B, Edleston A L W F, et al: chronic liverdisease developing after outbreak of HBsAg-negative hepatitis inhaemodialysis unit. Lancet, 2:886 (1975).

7. Mosley J W, Redeker A G, Feinstone S M, et al: Multiple hepatitisviruses in multiple attacks of acute viral hepatitis. N Engl J Med,296:75 (1977).

8. Dienstag J L, Purcell R H: Epidemiology of hepatitis non-A, non-B.Rush-Presbyterian-St. Luke's Med Bull, 15:104 (1976).

9. Aach R D, Szmuness W, Mosley J W, et al: Serum alanineaminotransferase of donors in relation to the risk of non-A, non-Bhepatitis in recipients: the Transfusion-Transmitted Viruses Study. NEngl J Med 304:989 (1981).

10. Hollinger F B, Mosley J W, Szmuness W, et al: "Non-A, non-Bhepatitis following blood transfusion: risk factors associated withdonor characteristics" in Viral Hepatitis: 1981 International Symposium,Szmuness W, Alter H J, Maynard J E, (eds), Philadelphia: FranklinInstitute Press, p361 (1982).

11. Alter H J, Purcell R H, Holland P V, Alling D W, Koziol D E: Donortransaminase and recipient hepatitis: impact on blood transfusionservices. JAMA, 246:630 (1981).

12. Stevens C E, Aach R D, Hollinger F B, et al: Hepatitis B virusantibody in blood donors and the occurrence of non-A, non-B hepatitis intransfusion recipients. Ann Int Med, 101:733 (1984).

13. Aach R D, Szmuness W, Mosley J W, et al: Serum alanineaminotransferase of donors in relation to the risk of non-A, non-Bhepatitis in recipients: The Transfusion-Transmitted virus Study. N EnglJ Med. 304:989 (1981).

14. Koziol D E, Holland P V, Alling D W, et al: Antibody to hepatitis Bcore antigen as a paradoxical marker for non-A, non-B hepatitis agentsin donated blood. Ann Int Med, 104:488 (1986).

15. Sugg U, Schenzle D, Hess G: Antibodies to hepatitis B core antigenin blood donors screened for alanine aminotransferase level andhepatitis non-A, non-B in recipients. Transfusion, 28:386 (1988).

16. Choo Q-L, Kuo G, Weiner A, et al: Isolation of a cDNA clone derivedfrom a blood-borne non-A, non-B viral hepatitis genome. Science, 244:359(1989).

17. Kuo G, Choo Q-L, Alter H J, et al: An assay for circulatingantibodies to a major etiologic virus of human non-A, non-B hepatitis.Science, 244:362 (1989).

18. Houghton M, Choo Q-L, Kuo G: NANBV diagnostics and vaccines. EPO0318218AT, 1989

19. Wang C Y: Synthetic-peptide-based immunodiagnosis of retrovirusinfections: current status and future prospects. In: Synthetic Peptidesin Biotechnology, A. Mizrahi (ed), Adv in Biotechnological Processes,10:131 (1988).

20. Wang J G, Steel S, Wisniewolski R, Wang C Y: Detection of antibodiesto HTLV-III using a synthetic peptide of 21 amino acid residuescorresponding to a highly antigenic segment of gp41 envelope protein.Proc Natl Acad Sci U.S.A., 83:6159 (1986).

21. Wang C Y: European Patent Application Publication: EPO 0328403(1989). Synthetic peptides related to the HIV-gp120 env. protein, andtheir use.

22. Wang C Y, Wang J G: U.S. Pat. No. 4,879,212 (1989). Peptidecomposition and method for the detection of antibodies to HTLV-III.

23. Wang C Y, Wang J G: U.S. Pat. No. 4,735,896 (1988). Syntheticpeptide and process of using same for the detection and diagnosis ofAIDS and pre-AIDS conditions.

24. Wang C Y, Wang J G, Walters D W: U.S. Pat. 4,833,071 (1989). Peptidecomposition as antigen for detection of antibodies to HTLV-1, as avaccine for ATL, and methods therefor.

25. Wang C Y: U.S. Ser. No. 07/297635. Synthetic peptide compositionswith immunoreactivities to antibodies to HTLV.

26. UBI-OLYMPUS HIV-1 EIA Product Insert. Jun. 1, 1989. License No.1079, approved by US FDA.

27. Okamoto, H., Okada, S., Sugiyama, S., Yotsumoto, S., Tanaka, T.,Yoshizawa, H., Tsuda, F., Miyakawa, Y., and Mayumi, M.: The 5' TerminalSequence of the Hepatitis C virus genome, Jpn. J. Exp Med. 1990 (inpress).

28. Schlesinger, S., and Schlesinger, M., (Ed.) The Togaviridae andFlaviviridae. In: The Viruses. Plenum Press, New York, 1986.

29. Kubo, Y., Takeuchi, K., et al: cDNA fragment of Hepatitis C virusisolated from an implicated donor of post-transfusion Non-A, Non-BHepatitis in Japan. Nucleu Acid Res. 17:10367-10372 (1989).

BRIEF DESCRIPTION OF THE INVENTION

According to the present invention, a series of synthetic peptidesrepresenting immunodominant regions of the hepatitis C virus (HCV)proteins, each arranged in a specific sequence, has been identified andmade by solid phase peptide synthesis. These peptides have been found tobe useful in a highly sensitive and accurate method for the earlydetection of antibodies to HCV in sera and body fluids and the diagnosisof non-A non-B hepatitis (NANBH). Because of their highimmunoreactivity, it is expected that these peptides are also useful instimulating production of antibodies to HCV in healthy mammals such asBalb/C mice, and in a vaccine composition to prevent HCV or NANBHVinfection.

According to the present invention, a peptide composition useful for thedetection of antibodies to HCV and diagnosis of NANBH comprises apeptide selected from the group of peptides with the followingsequences: ##STR1## wherein X is --OH or --NH₂, and analogues, segments,mixtures, combinations, conjugates and polymers thereof.

The amino acids in this application are abbreviated as shown hereinbelow:

A=Ala=alanine,

R=Arg=arginine,

D=Asp=Aspartic acid,

N=Asn=asparagine,

Q=Gln=glutamine,

E=Glu=glutamic acid,

L=Leu=leucine,

K=Lys=lysine,

H=His=histidine,

T=Thr=threonine,

G=Gly=glycine,

I=Ile=isoleucine,

F=Phe=phenylalanine,

S=Ser=serine,

W=Trp=tryptophan,

Y=Tyr=tyrosine,

V=Val=valine,

C=Cys=cysteine,

P=Pro=proline

An example of a combination is:Cys-Val-Val-Ile-Val-Gly-Arg-Val-Val-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Xwherein X is --OH or --NH₂. An example of a segment of Peptide II is:Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-X wherein X is --OHor --NH₂ (IIF). An example of a segment of Peptide III is:Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Xwherein X is --OH or --NH₂ (IIID). An example of a segment of Peptide IXis Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Trp-Ala-Gly-Trp-Leu-Leu-Ser-Pro-Arg-Gly-Ser-Arg-Pro-Ser-Trp-Gly-Pro-Thr-Asp-Pro-Arg-Arg-Arg-Ser-Arg-Asn-Leu- Gly-X(IXC).

The present invention also includes a highly sensitive and accuratemethod of detecting antibodies to HCV in body fluids and of diagnosingNANBH comprises the following steps:

A. Preparing a peptide composition comprising a peptide selected fromthe group having the following amino acid sequences: ##STR2## wherein Xis --OH or --NH₂, and analogues, segments, mixtures, combinations,conjugates and polymers thereof; and

B. Using an effective amount of the peptide composition as the antigenin an immunoassay procedure.

Further, according to the present invention, the peptides by themselves,or when coupled to a protein or a polymeric carrier of homo or heterodimers or higher oligomers by use of homo or hetero functionalmultivalent cross linking reagents, or when directly synthesized andconjugated to a branching polyvalent lysine resin, can be used to elicitthe production of antibodies to HCV in healthy mammals, includinghumans.

The method comprises introducing an effective amount of the peptidecomposition containing each of the individual peptides, analogues orsegments or a mixture or a combination thereof, or in a polymeric form,into the body of a healthy mammal by intraperitoneal or subcutaneousinjection.

Vaccines containing the peptides according to the present invention asthe key immunogen may also be prepared. It is expected that such vaccinecompositions may be useful to prevent HCV infection or NANBH.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-1, 1-2, 1-3 and 1-4 show the amino acid sequences of theimmunodominant region of a HCV non structural protein and preciselydelineates the amino acid residues (underlined to show - - - marginal,₋₋ moderate, and strong) that contribute to the immunoreactivities ofthese HCV peptides with four representative HCV antibody positive sera.The immunoreactivities were measured as absorbance at 492 nm by an EIAprocedure.

FIGS. 2-1 and 2-2 are comparisons of the signal to cutoff ratio betweenthe peptide based HCV-EIA employing only the non-structural proteinsequence derived Peptide IIG of the present invention and that of therecombinant SOD/HCV C-100-3 protein based HCV-EIA. In FIG. 2-1 awell-characterized HCV antibody positive control at various serumdilutions was used as the test sample. In FIG. 2-2 a panel of serumspecimens derived from serial bleedings of a single individual spanninga period of sero-conversion to anti-HCV reactivity were used as samples.

FIGS. 3-1 and 3-2 depict the frequency distribution of the HCV-EIApositivity, using Peptide IIG, represented by the signal to cutoffratios obtained with 264 normal serum and 264 normal plasma specimensfrom commercial sources. The mean s/c ratios for the negative (n=250)and screened out positive (i.e. n=14) serum specimens are 0.034 and7.202 respectively; and for the negative (n=255) and positive (n=9)normal plasma specimens the mean s/c ratios are 0.084 and 7.089respectively.

FIG. 4 is a histogram depicting the immunoreactivities of Peptide IIGwith sera from individuals: (a) positive for HBsAg, (n=50); (b) positivefor antibodies to HBc protein, (n=39); (c) with elevated (>100 I.U./L)alanine aminotransferase (ALT) enzyme activity, (n=174); (d) positivefor antibodies to retroviruses HIV-1 (n=100), HIV-2 (n=10), HTLV-I/II(n=14); all asymptomatic, (total n=124); (e) with AIDS, ARC (N=200) orATL (n=170) disease, (total n=270); and (f) with autoimmune disease(n=20).

FIG. 5 provides a comparison between EIA results using the Peptides IIFand IIID of the present invention and recombinant SOD/HCV C-100-3 asrepresented by their respective s/c ratios on a panel of repeatablyreactive specimens (n=23) obtained from a random donor population.

FIG. 6 provides a comparison between a passive hemagglutination assay(PHA), using Peptide IIG, and the recombinant SOD/HCV C-100-3 EIA asrepresented by their respective P/C and s/c ratios for a panel ofSOD/HCV C-100-3 HCV EIA repeatably reactive specimens (n=20) obtainedfrom a random donor population. For results obtained by the PHA, theagglutination pattern is quantitated by a specially designed opticalreading instrument (manufactured by Olympus Corporation) where a P/Cratio of larger than 20 is considered negative whereas a P/C ratio ofless than 20 is considered positive.

FIG. 7-1 provides a study of serum samples collected over a ten yearperiod of time from a NANBH patient who sero-converted after receivingHCV infected blood. The samples were tested by two EIA formatsdesignated as A (coated with Peptides IIF and IIID at 5 ug/mL each) andB (coated with Peptides IIF, IIID and V at 5 ug/mL each) for comparison.The serum samples were provided by Dr. H. Alter of NIH.

FIG. 7-2 provides a kinetic study with serum samples, kindly provided byDr. C. Stevens of New York Blood Center, from a hemodialysis patient whosero-converted and contracted NANBH. These were tested by EIA format B(coated with peptides IIF, IIID and V at 5 ug/mL each).

FIG. 7-3 provides a second kinetic study with serum samples, kindlyprovided by Dr. D. Bradley of Center for Disease Control, from achimpanzee which sero-converted after being inoculated with awell-characterized strain of HCV and contracted NANBH, also tested byEIA format B.

FIGS. 8-1 and 8-2 depict the signal/cutoff ratio frequency distributionof both negative and positive serum specimens by a HCV-EIA format B. Theresults were obtained using 2035 low risk random blood donor specimensscreen tested in a blood bank setting.

FIG. 9 illustrates the inhibition by Peptide IV (an analogue) of bindingof HCV specific antibodies to plates coated with peptides IID and IIIFat 5 ug/mL each at various Peptide IV concentrations.

FIG. 10 provides a comparison between the peptide based HCV EIA (coatedwith Peptide IIH and V at 10 and 5 ug/mL respectively) and recombinantprotein based HCV EIA using samples from 74 hemodialysis patients,kindly provided by investigators at the Japanese National Institute ofHealth.

FIGS. 11-1, and 11-2 show the amino acid sequences of an immunodominantregion of the postulated HCV structural (core or nucleocapsid) proteinand precisely delineates the amino acid residues that contribute to theimmunoreactivities of these HCV peptides with four representative HCVantibody positive sera (Samples 1-4). The immunoreactivities weremeasured as absorbance at 492 nm by an EIA procedure.

FIG. 12-1, 12-2 and 12-3 are histograms depicting the frequencydistribution of HCV positivity in 221 sera from individuals: (a) withAIDS, ARC (n=63); (b) positive for HBsAg, (n=50); (c) positive forantibodies to HBc protein, (n=22); (d) with elevated (>100 I.U./L)alanine aminotransferase (ALT) enzyme activity, (n=86) tested usingthree HCV EIA formats using Peptides IIH, V and VIIIE at 5, 3, and 2ug/mL respectively (Format C); Peptides VIIIE, and IXD at 2 and 2 ug/mLeach (Format D), and Peptides IIH and V at 5 and 3 ug/mL each (FormatA).

FIGS. 13-1, 13-2, 13-3, 13-4, 13-5, and 13-6 depict the signal to cutoffratio frequency distribution of HCV positivity in low risk random donorspecimens using three HCV-EIA Formats, A (13-1 and 13 2), C (13-3 and13-4), and D (13-5 and 13-6). The results were screen tested in a bloodbank setting.

FIG. 14-1 provides a study of serum samples collected over a ten yearperiod of time from a NANBH patient who sero-converted after receivingHCV infected blood. The samples were tested by a third EIA formatdesignated as C (coated with Peptides IIH, V, and VIIIE at 5, 3 and 2ug/mL respectively) in comparison to two other EIA formats (designatedas A and B.)

FIG. 14-2 provides another kinetic study with serum samples, kindlyprovided by Dr. D. Bradley of Center for Diseases Control, from achimpanzee which sero-converted after being inoculated with awell-characterized strain of HCV and contracted NANBH. These sampleswere tested by the HCV EIA Format C, in comparison to a RIA using rDNAbased HCV C-100 protein as the antigen. The ALT levels are alsoindicated with each bleed as a reference parameter.

FIGS. 15-1 and 15-2 both provide a side-by side data comparison via x-yplots with samples from hemodialysis patients, kindly provided byinvestigators at the Japanese National Institute of Health. The resultswere obtained by using the peptide based HCV EIA Format C (coated withpeptides derived from both the structural and non-structural proteinscontaining IIH, V and VIIIE at 5, 3, and 2 ug/mL respectively), HCV EIAFormat A (coated with peptides derived from the nonstructural proteinregion containing IIH and V at 5 and 3 ug/mL respectively). and therecombinant HCV C-100 protein based EIA.

The amino acids in the drawings and tables are abbreviated using the artaccepted single letter codes as follows:

A=Ala=alanine,

R=Arg=arginine,

D=Asp=aspartic acid,

N=Asn=asparagine,

Q=Gln=glutamine,

E=Glu=glutamic acid,

L=Leu=leucine,

K=Lys=lysine,

H=His=histidine,

T=Thr=threonine,

G=Gly=glycine,

I=Ile=isoleucine,

F=Phe=phenylalanine,

S=Ser=serine,

W=Trp=tryptophan,

Y=Tyr=tyrosine,

V=Val=valine,

C=Cys=cysteine,

P=Pro=proline,

M=Met=methionine

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, three peptides and theirsegments have been chemically synthesized for the detection ofantibodies to HCV in body fluids, the diagnosis of NANBH, and for thevaccination of healthy mammals by stimulating the production ofantibodies to HCV. These peptides are arranged in the followingsequences: ##STR3## wherein X is --OH or --NH₂.

These peptides may comprise combinations or segments, i.e. longer orshorter peptide chains by having more amino acids added to the terminalamino acids, or by amino acids removed from either terminal end.

These peptides may also comprise analogues to accommodatestrain-to-strain variations among different isolates of HCV. HCV isindicated to have frequent mutations. Therefore, it is expected thatvariant strains, such as J-1 and J-4 (see reference #27), exist.Adjustments for conservative substitutions and selection among thealternatives where non-conservative substitutions are involved, may bemade in the prescribed sequences. It is expected that as long as thepeptide's immunoreactivity recognizable by the antibodies to HCV ispreserved, analogues of the synthetic peptide may also comprisesubstitutions, insertions and/or deletions of the recited amino acids ofthe above sequence.

These peptides may also comprise conjugates, i.e., they may be coupledto carrier proteins such as bovine serum albumin (BSA) or human serumalbumin (HSA). Furthermore, these peptides may comprise polymers, i.e.,they may be synthesized on a polymeric resin, such as a branchingoctameric lysine resin.

The amino acid sequences of the polypeptides useful as test reagents forthe detection of antibodies to HCV in body fluids and diagnosis of NANBHare selected to correspond to a partial segment of the amino acidsequence of the HCV proteins: a non-structural protein designated as HCVC-100(18), and a structural protein such as the core (nucleocapsid)protein (27).

In selecting regions of the HCV protein for epitope analysis, peptidesin the 40 mer size range with amino acid sequences covering the completeHCV C-100 protein and the postulated core protein were synthesized.These were tested for their immunoreactivity with serum from a patientpositively diagnosed with HCV infection. Six overlapping peptides fromthe HCV C-100 protein region designated as I, II, III, IV, V and VI andtwo adjacent peptides form the postulated core protein region designatedas VIII and IX were identified to have specific immunoreactivity withthe positive HCV serum. Another peptide vII and its fragments,C-terminal to this immunodominant region, was also found to havemoderate immunoreactivity with a sub population of HCV positive sera.See Example 12. Peptide IIH, another analogue of Peptide II, with fiveadditional amino acids to the N-terminus has been found to be highlyimmunogenic and contains an additional epitope recognizable byantibodies in sera from patients with acute phase NANBHv infection (withelevated ALT levels). The amino acid sequences of the peptides are asfollows: ##STR4##

The six peptides I, II, III, IV, V and VI span a region of 90 aminoacids: ##STR5## and were found to have specific immunoreactivity withthe positive control serum. Table 1 shows the amino acid sequence ofthis immunodominant region of the HCV protein, and presents the aminoacid sequence of the six chemically synthesized peptides, designated asI to VI and segments (A to H) thereof.

Another two peptides (VIII and IX) spanning a region of 119 amino acidslocated inside the 5' terminal of the postulated HCV core protein:##STR6## were found to have specific immunoreactivity with arepresentative panel of well-characterized HCV antibody positive sera.

Table 7 shows the amino acid sequence of this immunodominant region ofthe postulated HCV core protein, and presents the amino acid sequence ofthe ten chemically synthesized peptides. They were designated, asPeptides VIII and IX with segments (A to D) thereof. Each of thesepeptides was coated at 5 ug/mL in a 10 mM sodium bicarbonate buffer (pH9.5) onto polystyrene microwell plates and tested in a three step 45minute enzyme immunoassay procedure, described hereinbelow, with a panelof HCV antibody positive sera, each selected as representative of aparticular clinical population, at various serum dilutions.

    TABLE 1      CHARACTERIZATION OF THE IMMUNODOMINANT REGION OF THE HCV SOD-C100     FUSION POLYPEPTIDE:    RELATIVE (%) CV,VIVGR, VVLSG,KPAII,PDREV,LYREF,DEM     EE,CSQHL,PYIEQ,GMMLA,EQFKQ,KALGL,LQTAS,RQAEV,IAPAV, QTNWQ,KLETF,WAKHM,WNF      IMMUNOREACTIVITY       IA  GL,LQTAS,RQAEV,IAP  3.0 IB  KQ,KALGL,LQTAS,RQAEV,IAP  10.3 IC      ##STR7##       23.9 ID  EQ,GMMLA,EQFKQ,KALGL,LQTAS,RQAEV,IAP  24.6 IE      ##STR8##       38.2 IF      ##STR9##       45.6 IIA  GMMLA,EQFKQ,KALGL  3.1 IIB      ##STR10##       24.3 IIC      ##STR11##       41.7 IID  DEMEE,CSQHL,PYIEQ,GMMLA,EQFKQ,KALGL  44.9 IIE      ##STR12##       57 IIF      ##STR13##       99 IIG  II,PDREV,LYREF,DEMEE,CSQHL,PYIEQ,GMMLA,EQFKQ,KALGL  93.2 IIH     SG,KPAII,PDREV,LYREF,DEMEE,CSQHL,PYIEQ,GMMLA,EQFKQ,KALGL  101 IIIA     EF,DEMEE,CSQHL,PYI  4.9 IIIB      ##STR14##       26.3 IIIC      ##STR15##       85 IIID      ##STR16##       100 IIIE GR, VVLSG,KPAII,PDREV,LYREF,DEMEE,CSQHL,PYI  99 IIIF CV,VIVGR,      VVLSG,KPAII,PDREV,LYREF,DEMEE,CSQHL,PYI  95 IV  SG,KPAII,PDREV,LYREF,DEM     EE,CSQHL,PYIEQ,GMMLA,EQF  93.8 VIA  AS,RQAEV,IAPAV, QTNWQ,KLET 3.9 VIB      ##STR17##      QTNWQ,KLET 43.6 VIC  KQ,KALGL,LQTAS,RQAEV,IAPAV, QTNWQ,KLET 44.0 VID     LA,EQFKQ,KALGL,LQTAS,RQAEV,IAPAV, QTNWQ,KLET 46.0 VIE      ##STR18##      QTNWQ,KLET 54.8 VA  AV, QTNWQ,KLETF,WAKHM,WNF 1.3 VB      ##STR19##      QTNWQ,KLETF,WAKHM,WNF 17.8 VC      ##STR20##      QTNWQ,KLETF,WAKHM,WNF 23.4 VD  GL,LQTAS,RQAEV,IAPAV, QTNWQ,KLETF,WAKHM,W     NF 32.3 V      ##STR21##      QTNWQ,KLETF,WAKHM,WNF 93.9     ##STR22##

    TABLE 7      Identification and characterization of An Immunodominant Region in     HCV's Structural Proteins, based on the predicted amino acid sequence     derived from the structural genes of two HCV genomes (27), by serological      validation with a combination of synthetic peptides (VIIIA-E and     IXA-E):                     J-1J-4     ##STR23##      ##STR24##      ##STR25##      ##STR26##      ##STR27##      ##STR28##      ##STR29##      ##STR30##      ##STR31##      ##STR32##      ##STR33##      ##STR34##      ##STR35##                    %               Relat Peptide              Immuno- Code                react       VIIIE STIP, KPQRK, TKRNT, NRRPQ, DVKFP, GGGQI, VGGVY, LLPRR, GPRLG,     VRATR, KTSER, SQPRG, RR 98.6 VIIID   TKRNT, NRRPQ, DVKFP, GGGQI, VGGVY,     LLPRR, GPRLG, VRATR, KTSER SQPRG, RR 84.1 VIIIC     DVKFP, GGGQI, VGGVY,     LLPRR, GPRLG, VRATR, KTSER, SQPRG, RR 70.2 VIIIB       VGGVY, LLPRR,     GPRLG, VRATR, KTSER, SQPRG, RR 54.8 VIIIA         GPRLG, VRATR, KTSER,     SQPRG, RR 27.5 IXE            G, RRQPI, 49.5 IXD            I, 58.1 IXC                 57.1 IXB              48.4 IXA              23.8       Identification and characterization of An Immunodominant Region in     HCV's Structural Proteins, based on the predicted amino acid sequence     derived from the structural genes of two HCV genomes (27), by serological      validation with a combination of synthetic peptides (VIIIA-E and     IXA-E):                   J-1J-4     ##STR36##      ##STR37##      ##STR38##      ##STR39##      ##STR40##      ##STR41##      ##STR42##      ##STR43##      ##STR44##      ##STR45##      ##STR46##                  %             Relat Peptide            Immuno- Code        react       VIIIE            98.6 VIIID            84.1 VIIIC            70.2     VIIIB            54.8 VIIIA            27.5 IXE PKVRR, PEGRT, WAQPG,     YPWPL, YGNEG, CGWAG, WLLSP, RGSRP, SWGPT, DPRRR, SRNLG 49.5 IXD PKVRR,     PEGRT, WAQPG, YPWPL, YGNEG, CGWAG, WLLSP, RGSRP, SWGPT, DPRRR, SRNLG     58.1 IXC T, WAQPG, YPWPL, YGNEG, CGWAG, WLLSP, RGSRP, SWGPT, DPRRR,     SRNLG 57.1 IXB   L, YGNEG, CGWAG, WLLSP, RGSRP, SWGPT, DPRRR, SRNLG 48.4     IXA       WLLSP, RGSRP, SWGPT, DPRRR, SRNLG 23.8      Calculations based on the overall EIA absorbance of all positive sera     yielded an array of immunoreactivity indices represented as % relative     immunoreactivity for each of the synthetic HCV peptides. Three peptides,     designated as IIF, IIH and IIID, being 40 mer, 47 mer and 30 mer in size,     with the following amino acid sequence respectively:     ##STR47##     were found to have the highest immunoreactivity with the sera panel. The     relative (%) immunoreactivity for each of the 40 HCV peptides listed in     Tables 1 and 7, as a result of this extensive epitope mapping study,     provides a basis for the delineation of several clusters of amino acid     residues (as underlined), each in a prescribed sequence, that are involved     in or relevant to the antigenic configuration of the HCV peptides. Two     peptides, designated as VIIIE and IXD being 61 mer and 56 mer in size are     respectively located within the HCV structural core protein region with     the following amino acid sequences:     ##STR48##     Peptides XIIIE and IXD were also found to have the highest reactivity in     this region.

Assays for antibodies to HCV based upon chemically synthesized peptidesshow several advantages over assays utilizing biologic basedimmunoadsorbents. The peptides can easily be synthesized in gramquantities by using automated solid-phase methods, thus providing areproducible antigen of high integrity with consistent yields. Thepresence of other antigens from biological systems precludes suchreproducibility. More importantly, non-specific reactivities seen inuninfected individuals are likely to be due to the heterogeneity of thepreparations used for assay. This is particularly true for assays usingbiologically based immunoadsorbents. In these processes, the hostantigens are frequently co-purified with the desired viral protein(s).Antibodies to these contaminating antigens are frequently found innormal individuals, thus resulting in false-positive results.

The assay of the present invention clearly minimizes such false-positivereactions as encountered in the other assay systems and, at the sametime, shows a high sensitivity to truly positive sera by thesubstantially increased signal-to-noise ratio. This increased signal-tonoise ratio probably resulted from the purity of the immunoadsorbent.The assay of the present invention is also highly specific, in that themean S/C ratios for HCV carriers are about 80-200 times the mean S/C ofthose of the non-infected individuals. For a representative example, seeFIGS. 3-1 and 3-2.

The peptides useful as solid phase immunoadsorbents for the detection ofantibodies to HCV were synthesized by the "classical" Merrifield methodof solid phase peptide synthesis using slide chain protected t-Boc-aminoacids to correspond to the following amino acid sequences: ##STR49##wherein X is --NH₂.

Other analogues, segments and combinations of these peptides may beprepared by varying the amino acid sequences either by adding,subtracting, substituting, or deleting desired t-Boc-amino acid(s).

Following completion of assembly of the desired blocked peptide on theresin, the peptide-resin is treated with anhydrous hydrofluoric acid tocleave the peptide from the resin. Functional groups of amino acidswhich are blocked during synthesis by benzyl-derived blocking groups arealso cleaved from the peptide simultaneously. The free peptide is thenanalyzed and purified by high performance liquid chromatography (HPLC)and characterized biochemically by amino acid analysis.

Longer peptides with more than about 50 amino acids may also be preparedconveniently using well known recombinant methods. The known nucleicacids codons for each of the amino acids in the peptide may be utilizedand synthetic genes encoding such peptides constructed. The syntheticgene may be inserted into vector constructs by known techniques, clonedand transfected into host cells, such as E. coli, or yeast. The secretedpolypeptide may then be processed and purified according to knownprocedures. The peptides synthesized according to the above describedprocedures are highly reactive with antibodies to HCV and can be used asa highly sensitive and specific immunoadsorbent for the detection ofantibodies to HCV.

FIGS. 1-1, 1-2, 1-3 and 1-4 and FIGS. 11-1 and 11-2 show the amino acidsequences of the immunodominant regions of HCV proteins, both structuraland non-structural, and precisely delineates, in the case of thenon-structural protein HCV C-100 region, the underlined amino acidresidues that contribute (- - - marginally, ₋₋ moderately, or strongly)to the immunoreactivities, measured at A492 nm by a peptide based EIAprocedure of these HCV peptides with four representative HCV antibodypositive sera.

The peptide based EIA procedure used to measure the immunoreactivity ofeach peptide is as follows. 100 uL per well of each of the peptides wascoated at 5 ug/mL in a pH 9.5 sodium bicarbonate buffer (10 mM) onto apolystyrene microwell plate and the microwell plate was incubated at 37°C. for about an hour, washed and dried. The test serum samples werediluted with PBS containing normal goat serum, gelatin and TWEEN 20. 200uL of the test serum sample solution was added to each well and allowedto react for 15 mins. at 37° C. The wells were washed, enzyme labelledantibodies were used to bind the HCV-antibody-peptide complex, and theplate was incubated for another 15 min. A color developer, e.g.orthophenylenediamine (OPD), was then added. The reaction was stoppedafter 15 min by the addition of 50 uL 1.0M H₂ SO₄, and the absorbance ofthe reaction mixture was read at 492 nm with an ELISA reader.

As demonstrated in FIG. 1-1, serum sample 1 has little reactivity withPeptide IA and IB. However, its reactivity with Peptide IC increasessignificantly, followed by a marginal increase with Peptide ID, andadditional increases with Peptides IE and IF. This indicates that in theHCV Peptide I series, two clusters of amino acid residues, namelyLeu-Ala-Glu-Gln-Phe and LAEQF and His-Leu-Pro-Tyr-Ile and HLPYI and, arecontributing to the antigenic determinant(s) of the HCV Peptide I.Similarly, a cluster of residues namelyGlu-Glu-Cys-Ser-Glu-His-Leu-Pro-Tyr-Ile and EECSQHLPYI and iscontributing to the immunoreactivity of the HCV Peptide II series;another cluster of residues namelySer-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg and SGKPAIIPDR and iscontributing to the immunoreactivity of HCV Peptide III series and twoclusters of residues, namely Gly-Leu-Leu-Glu-Thr and GLLQT and andGlu-Val-Ile-Ala-Pro and EVIAP and are contributing to theimmunoreactivity by HCV Peptides IV and V series. As shown on the bottomof FIG. 1-1, a total of six spaced clusters of amino acid residuesrepresenting discontinuous epitopes in this immunodominant region of theHCV protein are identified as contributing to the specific HCVimmunoreactivity with serum sample 1.

FIG. 1-2 illustrates an immunoreactivity profile for serum sample 2 whentested on a total of 31 overlapping peptides in the HCV Peptide I, II,III, IV, V and VI series. There is a clear difference between theimmunoreactivity profiles of serum samples 1 and 2. The immunodominantepitope, as marked by residues Ser-Gly-Lys-Pro-Ala and SGKPA andIle-Ile-Pro-Pro-Asp-Arg-Glu-Val and IIPDREV and, is located towards theN-terminus of the region.

FIG. 1-3 illustrates an immunoreactivity profile for serum 3 when testedon the same 31 HCV peptide panel. Through this extensive epitope mappinganalysis, serum sample 3 was found to have a similar immunoreactivityprofile to that of serum sample 2.

FIG. 1-4 illustrates an immunoreactivity profile for serum sample 4which differs significantly from that of sample 2 and 3, whilemaintaining some similarity to that of sample 1.

In summary, epitope mapping analysis conducted with a series of 31overlapping peptides covering an immunodominant region of the HCVnon-structural protein, which spans a total of 90 amino acid residues asillustrated in Table 1, and an immunodominant region of the HCVstructural core protein, which spans a total of 119 amino acid residuesas illustrated in Table 7, reveals a varying degree of immunoreactivityamong different HCV antibody positive samples and these HCV peptides.Based on overall EIA absorbance readings obtained with a panel of eightHCV positive sera with each of these 31 HCV peptides (Table 2), arelative (%) immunoreactivity index is established for each of thepeptides and several clusters of amino acid residues are identified ascontributing strongly, as in the cases ofIle-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg and Glu-Val-Ile-Ala-Pro;moderately, as in the cases of Ser-Gly-Lys-Pro-Ala,Glu-Val-Leu-Tyr-Arg-Glu-Phe,Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly; andLeu-Ala-Glu-Gln-Phe-Lys-Gln; or marginally, as in the case ofLys-Gln-Lys-Ala-Leu, to the HCV immunoreactivity.

Similarly, the relative immunoreactivity of Peptide VIII and IX andtheir analogue-segments are presented in Table 7.

                                      TABLE 2                                     __________________________________________________________________________    HCV Peptide Segments                                                          __________________________________________________________________________               Specimens                                                                     I                        II                                                   A   B   C   D   E   F   A   B   C   D   E   F   G                  __________________________________________________________________________    Blank      0.041                                                                             0.041                                                                             0.041                                                                             0.041                                                                             0.041                                                                             0.041                                                                             0.041                                                                             0.040                                                                             0.040                                                                             0.041                                                                             0.041                                                                             0.041                                                                             0.044              NRC        0.047                                                                             0.050                                                                             0.049                                                                             0.049                                                                             0.052                                                                             0.055                                                                             0.045                                                                             0.044                                                                             0.048                                                                             0.046                                                                             0.048                                                                             0.088                                                                             0.074              WRC        0.040                                                                             0.048                                                                             0.077                                                                             0.084                                                                             0.155                                                                             0.221                                                                             0.040                                                                             0.042                                                                             0.220                                                                             0.153                                                                             0.241                                                                             0.399                                                                             0.365              SRC        0.049                                                                             0.055                                                                             0.330                                                                             0.383                                                                             0.828                                                                             1.175                                                                             0.043                                                                             0.093                                                                             1.188                                                                             0.963                                                                             1.279                                                                             1.832                                                                             1.672              1          0.066                                                                             0.218                                                                             1.925                                                                             2.151                                                                             2.994                                                                             3.247                                                                             0.075                                                                             0.838                                                                             3.219                                                                             3.282                                                                             3.494                                                                             3.316                                                                             3.395              2          0.054                                                                             0.095                                                                             0.080                                                                             0.093                                                                             0.171                                                                             0.337                                                                             0.066                                                                             0.103                                                                             0.243                                                                             0.536                                                                             0.872                                                                             2.929                                                                             2.746              3          0.062                                                                             0.089                                                                             0.062                                                                             0.064                                                                             0.068                                                                             0.108                                                                             0.065                                                                             0.058                                                                             0.121                                                                             0.129                                                                             0.371                                                                             2.406                                                                             2.696              4          0.082                                                                             1.068                                                                             1.391                                                                             1.912                                                                             1.994                                                                             2.726                                                                             0.074                                                                             2.769                                                                             2.387                                                                             2.437                                                                             2.822                                                                             3.289                                                                             3.169              5          0.063                                                                             0.083                                                                             0.136                                                                             0.156                                                                             0.246                                                                             0.216                                                                             0.057                                                                             0.065                                                                             0.104                                                                             0.085                                                                             0.197                                                                             0.732                                                                             0.261              6          0.059                                                                             0.073                                                                             0.058                                                                             0.066                                                                             0.071                                                                             0.071                                                                             0.061                                                                             0.068                                                                             0.066                                                                             0.061                                                                             0.086                                                                             0.623                                                                             0.488              7          0.050                                                                             0.052                                                                             0.058                                                                             0.062                                                                             0.091                                                                             0.091                                                                             0.046                                                                             0.049                                                                             0.066                                                                             0.048                                                                             0.152                                                                             1.146                                                                             1.100              8          0.070                                                                             0.087                                                                             0.254                                                                             0.293                                                                             0.710                                                                             0.698                                                                             0.070                                                                             0.076                                                                             0.718                                                                             0.812                                                                             1.463                                                                             1.998                                                                             1.624              8     A492 nm                                                                            0.056                                                                             1.765                                                                             3.964                                                                             4.077                                                                             6.345                                                                             7.494                                                                             0.514                                                                             4.026                                                                             6.924                                                                             7.390                                                                             9.457                                                                             16.44                                                                             15.48              i = 1                                                                         % Relative 3.0 10.3                                                                              23.9                                                                              24.6                                                                              38.2                                                                              45.6                                                                              3.1 24.3                                                                              41.7                                                                              44.9                                                                              57  99  93.2               Immuno-                                                                       reactivity                                                                    __________________________________________________________________________                                            Specimens                                                                     III                                                                           A  B   C   D   E   F                  __________________________________________________________________________                                 Blank      0.040                                                                            0.046                                                                             0.040                                                                             0.045                                                                             0.040                                                                             0.043                                           NRC        0.44                                                                             0.048                                                                             0.061                                                                             0.073                                                                             0.071                                                                             0.070                                           WRC        0.046                                                                            0.043                                                                             0.192                                                                             0.243                                                                             0.269                                                                             0.222                                           SRC        0.046                                                                            0.074                                                                             1.081                                                                             1.260                                                                             1.379                                                                             1.127                                           1          0.289                                                                            0.527                                                                             3.245                                                                             4.057                                                                             3.545                                                                             3.613                                           2          0.191                                                                            0.316                                                                             2.715                                                                             2.941                                                                             3.053                                                                             2.984                                           3          0.066                                                                            0.085                                                                             2.407                                                                             2.612                                                                             2.566                                                                             2.624                                           4          0.064                                                                            2.864                                                                             3.096                                                                             3.221                                                                             3.319                                                                             3.220                                           5          0.056                                                                            0.126                                                                             0.588                                                                             0.657                                                                             0.700                                                                             0.522                                           6          0.054                                                                            0.075                                                                             0.458                                                                             0.623                                                                             0.641                                                                             0.489                                           7          0.045                                                                            0.273                                                                             0.863                                                                             1.577                                                                             1.669                                                                             1.505                                           8          0.058                                                                            0.101                                                                             0.655                                                                             0.894                                                                             0.937                                                                             0.820                                           8     A 492 nm                                                                           0.823                                                                            4.367                                                                             14.03                                                                             16.58                                                                             16.43                                                                             15.78                                           i = 1                                                                         % Relative 4.9                                                                              26.3                                                                              85  100 99  95                                              Immuno-                                                                       reactivity                                       __________________________________________________________________________

Based on the above-mentioned epitope mapping study, four representativeEIAs were configurated using Peptide IIG alone, a mixture of twoPeptides IIF and IIID, a mixture of IIF, IIID and V, or a mixture of IIHand V as the solid phase antigen.

FIGS. 2-1 and 2-2 depict the comparison, by signal to cutoff ratio,between the peptide based HCV-EIA employing Peptide IIG, at 5 ug/mLcoating concentration, and that of recombinant SOD/HCV C-100-3 proteinbased HCV-EIA. In FIG. 2-1, a well-characterized HCV antibody positivecontrol at various serum dilutions was used as the sample. In FIG. 2-2,a panel of serum specimens derived from serial bleedings of a singleindividual spanning a period of sero-conversion to anti-HCV reactivitywas used. Similar dilution titers and equal ability to identify date ofsero-conversion, the two parameters indicative of the sensitivity ofeach assay, are obtained with the synthetic peptide based EIA accordingto the present invention and rDNA HCV C-100 based EIA, except that thepeptide based assay according to the present invention is moresensitive, conferring a higher signal to cutoff ratio to its positivespecimens.

FIG. 3-1 and 3-2 depict the frequency distribution of the syntheticpeptide based HCV-EIA signal to cutoff ratios, using Peptide IIG at 5ug/mL as the coating concentration, obtained with 264 normal serum and264 normal plasma specimens from commercial sources. The mean s/c ratiosfor the negative (n=250) and screened out positive (i.e. n=14) serumspecimens are 0.034 and 7.202 respectively; for the negative (n=255) andpositive (n=9) normal plasma specimens the mean ratios are 0.084 and7.089 respectively. A sharp contrast between the screened out positivesand all the negatives is obtained with the peptide based HCV-EIA of thepresent invention.

Based on the high degree of sensitivity and specificity of the peptidecompositions according to the present invention in theirimmunoreactivities to antibodies to HCV, it is believed that the peptidecompositions according to the present invention may also be useful asvaccines to prevent NANBH, and as immunogens for the development of bothmonoclonal and polyclonal antibodies to HCV in mammals, includinghumans. The peptide compositions when coupled to a protein, orsynthesized on a polymeric carrier resin (e.g., an octameric branchinglysine resin) or when polymerized to homo or hetero dimers or higheroligomers by cysteine oxidation, induced disulfide cross linking, or byuse of homo or hetero functional multivalent cross linking reagents, canbe introduced to normal subjects to stimulate production of antibodiesto HCV in healthy mammals.

The advantages of using the peptides according to the present inventionare many.

Since the peptide compositions according to the present invention arenot derived biologically from the virus, there is no danger of exposingthe normal subjects who are to be vaccinated to the disease.

The peptides can be chemically synthesized easily. This means that thereis no involvement with the HCV at any time during the process of makingthe test reagent or the vaccine. Another problem which can be minimizedby the process of the present invention is the false positive resultscaused by the presence of antigenic materials from host cellsco-purified with the HCV fusion protein. Certain normal individuals haveantibodies to E. coli or yeast proteins which are cross reactive withthe antigenic materials from host cells. Sera from these normalindividuals may show a positive response in the immunoassays.

Further, with appropriate amino acid modifications or substitutions, itis expected that various peptide analogues based on the prescribed aminoacid sequence can be synthesized with properties giving rise to lowerbackground readings or better binding capacity to solid phases usefulfor HCV antibody screening assays.

Moreover, because the peptide compositions of the present invention aresynthetically prepared, the quality can be controlled and as a result,reproducibility of the test results can be assured. Also, since verysmall amounts of peptides are required for each test procedure, andbecause the expense of preparing the peptides is relatively low, thecost of screening body fluids for antibodies to HCV, diagnosis of NANBHinfection, or the preparation of a vaccine is relatively low.

The peptides prepared in accordance with the present invention can beused to detect HCV infection and diagnose NANBH by using them as thetest reagent in an enzyme-linked immunoadsorbent assay (ELISA), anenzyme immunodot assay, an agglutination based assay, or otherwell-known immunoassay devices. The preferred method is ELISA. The ELISAtechnique is exemplified in Examples 1, 2, 8-10, 12 and 14-18 and theagglutination based assay in Examples 3 and 4. The Examples are used toillustrate the present invention and are not to be used to limit thescope of the invention.

It is to be noted that in the following methods, 0.25% by weight ofglutaraldehyde may be added to the coating buffer to facilitate betterpeptide binding onto the plates or beads. Further, horseradishperoxidase (HRPO) conjugated mouse monoclonal anti-human IgG antibody orthe HRPO conjugated second antibodies from any other animal species maybe used in place of the HRPO-conjugated goat anti-human IgG as thesecond antibody tracer.

The gelatin used in these processes can include calf skin gelatin, pigskin gelatin, fish gelatin or any known available gelatin proteins, orbe replaced with albumin proteins.

EXAMPLE 1 Measurement of Relative (%) Immunoreactivity for syntheticpeptide covering an immunodominant region of the HCV protein C-100 by anEnzyme-Linked Immunosorbent Assay

Wells or 96-well plates were coated at 4° C. overnight (or 1 hour at 37°C.), with each of the thirty one peptides: IA to IF, IIA to IIH, IIIA toIIIF, IV, V, VIA to VIE (see Table 1) prepared as described at 5 ug/mLat 100 uL per well in 10 mM NaHCO₃ buffer, pH 9.5. The peptide coatedwells were then incubated with 250 uL of 3% by weight of gelatin in PBSat 37° C. for 1 hour to block non-specific protein binding sites,followed by three washes with PBS containing 0.05% by volume of TWEEN 20and then dried. The test specimens were diluted with PBS containing 20%by volume normal goat serum, 1% by weight gelatin and 0.05% by volumeTWEEN 20 at dilutions of 1:20 volume to volume, respectively. 200 uL ofthe diluted specimens were added to each of the wells and allowed toreact for 15 minutes at 37°.

The wells were then washed six times with 0.05% by volume TWEEN 20 inPBS in order to remove unbound antibodies. Horseradish peroxidaseconjugated goat anti-human IgG was used as a second antibody tracer tobind with the HCV antibody-peptide antigen complex formed in positivewells. 100 uL of peroxidase labeled goat anti-human IgG at a dilution of1:1800 in 1% by volume normal goat serum, 0.05% by volume TWEEN 20 inPBS was added to each well and incubated at 37° C. for another 15minutes.

The wells were washed six times with 0.05% by volume TWEEN 20 in PBS toremove unbound antibody and reacted with 100 uL of the substrate mixturecontaining 0.04% by weight orthophenylenediamine (OPD) and 0.12% byvolume hydrogen peroxide in sodium citrate buffer, pH 5.0.

This substrate mixture was used to detect the peroxidase label byforming a colored product. Reactions were stopped by the addition of 100uL of 1.0M H₂ SO₄ and the absorbance measured using an ELISA reader at492 nm (i.e. A₄₉₂). Assays were performed in singlet at one specimendilution (1:20) with a panel of eight representative HCV antibodypositive sera, along with the specimen diluent blank, non-reactive,weakly reactive and strongly reactive controls (NRC, WRC, SRC) all induplicates.

Results obtained from this study are shown in Table 2. According to theEIA absorbance readings at 492 nm (y axis) and the amino acid sequencesfor each of the corresponding HCV peptides (x axis), representativeimmunoreactivity profiles are plotted for four of the eight sera asshown in FIGS. 1-1 to 1-4. Relative (%) immunoreactivity index for eachof the 31 peptides is calculated using Peptide IIID as a reference basedon the total absorbance of eight sera at 492 nm (See Tables 1 and 2).FIG. 1 shows the amino acid sequences of the immunodominant regionaccording to data presented in Tables 1 and 2, and precisely delineatesthe amino acid residues (underlined) that contribute (- - - marginally,₋₋ moderately, and strongly) to the immunoreactivities.

In summary, epitope mapping analysis conducted with a series of 31overlapping peptides covering an immunodominant region of HCV, spanninga total of 90 amino acid residues as illustrated in Table 1, reveals avarying degree of immunoreactivities between different HCV antibodypositive samples and these HCV peptides. Based on this study, severaldiscontinuous epitopes are located within this immunodominant region.Contrary to what is speculated by the conventional wisdom, it is foundpreferably to have peptides with longer amino acid chains, ideallylonger than 20, synthesized in order to optimally present theseantigenic determinants to HCV antibodies.

Based on the above-mentioned epitope mapping study, four representativeEIAs using peptide IIG alone, or a mixture of Peptides IIF and IID, or amixture of IIF, IIID and V, or a mixture of IIH and V as the solid phaseantigen were configurated for the following efficacy studies asdemonstrated in Examples 2, 8, 9, 10 and 12.

EXAMPLE 2 Detection of Antibodies to HCV by an Enzyme-LinkedImmunosorbent Assay

Wells of 96-well plates were coated at 4° C. overnight (or for 1 hour at37° C.) with either Peptide IIG alone at a coating concentration of 0.5ug per well (designated as IIG EIA) or with a mixture of two PeptidesIIF and IIID (designated as IIF/IIID EIA) in a ratio by weight ofIIF:IIID=1:1 at 1 ug per well of the mixture in 100 uL 10 mM NaHCO₃buffer pH 9.5. The peptide coated wells were then incubated with 250 uLof 3% by weight of gelatin in PBS at 37° C. for 1 hour to blocknon-specific protein binding sites, followed by three more washes withPBS containing 0.05% by volume of TWEEN 20 and dried.

The test specimens were diluted with PBS containing 20% by volume normalgoat serum, 1% by weight gelatin and 0.05% by volume TWEEN 20 atdilutions of 1:20 volume to volume, respectively. 200 uL of the dilutedspecimens were added to each of the wells and allowed to react for 15minutes at 37°.

The wells were then washed six times with 0.05% by volume TWEEN 20 inPBS in order to remove unbound antibodies. Horseradish peroxidaseconjugated goat anti-human IgG was used as a second antibody tracer tobind with the HCV antibody-peptide antigen complex formed in positivewells. 100 uL of peroxidase labeled goat anti-human IgG at a dilution of1:1800 in 1% by volume normal goat serum, 0.05% by volume TWEEN 20 inPBS was added to each well and incubated at 37° C. for another 15minutes.

The wells were washed six times with 0.05% by volume TWEEN 20 in PBS toremove unbound antibody and reacted with 100 uL of the substrate mixturecontaining 0.04% by weight orthophenylenediamine (OPD) and 0.12% byvolume hydrogen peroxide in sodium citrate buffer, pH 5.0. Thissubstrate mixture was used to detect the peroxidase label by forming acolored product. Reactions were stopped by the addition of 100 uL of1.0M H₂ SO₄ and the absorbance measured using an ELISA reader at 492 nm(i.e. A₄₉₂) Assays were performed in singlet at one specimen dilution(1:20) with all test specimens. Each plate run is accompanied by a panelof eight controls including the specimen diluent blank, negative, weakHCV reactive and strong HCV reactive controls, all in duplicate. Thestrongly reactive control was adjusted by diluting a HCV positive serumin the specimen dilution buffer at 1:300, which gave an absorbance valueat 492 nm of about 1.5 when performed in this standard 45 minute assayprocedure. A cutoff value is calculated based on the following formula:Cutoff=(0.1×SRC)+NRC. Both the raw absorbance (designated as signal) andthe ratio of signal to cutoff are recorded for all specimens analyzed.

The following groups of specimens were analyzed on the HCV peptide basedEIA according to the present invention, with the plates coated eitherwith 5 ug/mL of peptide IIG or a mixture containing 5 ug/mL IIF and 5ug/mL IIID:

(a) A well-characterized HCV antibody positive control based on serumdilutions; (on both IIG and IIF/IIID EIAs)

(b) a panel of serum specimens derived from serial bleedings of a singleindividual spanning a period of sero-conversion to anti-HCV reactivity;(on both IIG and IIF/IIID plates)

(c) 264 normal serum and 264 normal plasma specimens from commercialsources; (on IIG plates only)

(d) individuals positive for HBsAg, (n-30); (on both IIG and IIF/IIIDplates)

(e) individuals positive for antibodies to HBc protein, (n=39); (on bothIIG and IIF/IIID plates)

(f) individuals with elevated (>100 I.U./L) alanine aminotransferase(ALT) enzyme activity, (n=174); (on both IIG and IIF/IIID plates)

(g) individuals positive for antibodies to retroviruses HIV-1(n=100),HIV-2(n=10), HTLV-I/II(n=14); all asymptomatic, (total n=124); (on bothIIG and IIF/IIID plates)

(h) individuals with AIDS, ARC(n=200) or ATL (n=170) disease, (totaln=370); (on both IIG and IIF/IIID plates) and

(i) individuals with autoimmune disease (n=20). (on IIG plates only)

(j) recombinant SOD/HCV C-100-3 HCV-EIA repeatably reactive specimensobtained from a random donor population, (n=23). (on both IIG andIIF/IIID plates).

Results obtained from groups (a) and (b) are presented in FIGS. 2-1 and2-2 respectively (data obtained on IIG plates only), from group (c) inFIGS. 3-1 and 3-2; from groups (d) to (i) in FIG. 4, from group (j) inTable 3 and FIGS. 5 and 6.

In brief, as shown in FIGS. 2-1 and 2-2, a comparison, by signal tocutoff ratio, between the peptide based HCV-EIA of the present inventionemploying peptide IIG and that of recombinant SOD/HCV C-100-3 proteinbased HCV-EIA produced by Chiron/Ortho. Similar dilution titers andequal ability to identify date of sero-conversion, the two parametersindicative of each assay's sensitivity, are obtained for both assays.However, the assay according to the present invention is more sensitiveand confers a higher signal to cutoff ratio to its positive specimens.

                                      TABLE 3                                     __________________________________________________________________________    rDNA                              Peptide                                     SAMPLE                                                                              HCV  RPT                                                                              RPT                                                                              ALT Anti-HBc                                                                            OTHER  HCV-EIA                                     ID No.                                                                              S/C  S/C                                                                              S/C                                                                              (IU/L)                                                                            (S/C) POSITIVES                                                                            S/C                                         __________________________________________________________________________    1   161                                                                             5.33 5.56                                                                             5.56                                                                             36/56                                                                             2.10         11                                          2   280                                                                             5.76 5.56                                                                             5.56                                                                             78/56                                                                             0.07+ HBc, ALT                                                                             10                                          3   374                                                                             1.98 2.45                                                                             2.45                                                                             20/56                                                                             1.97         0.573                                       4   517                                                                             5.79 5.68                                                                             5.68                                                                             34/56                                                                             2.04         11                                          5   561                                                                             1.74 2.75                                                                             2.47                                                                             21/56                                                                             2.46         0.172                                       6   675                                                                             0.93 1.33                                                                             1.54                                                                             29/56                                                                             1.98         0.135                                       7   720                                                                             5.68 5.68                                                                             5.68                                                                             57/56                                                                             0.08+ HBc, ALT                                                                             13                                          8   773                                                                             5.56 5.88                                                                             5.88                                                                             86/56                                                                             2.07  HIV, ALT                                                                             8.625                                       9   797                                                                             3.79 4.35                                                                             4.29                                                                             74/56                                                                             0.38+ HBc, ALT                                                                             1.802                                       10  869                                                                             5.66 5.59                                                                             5.59                                                                             35/56                                                                             2.45         9.755                                       11  873                                                                             5.66 5.59                                                                             5.59                                                                             26/56                                                                             2.34         1.189                                       12 1003                                                                             1.63 1.24                                                                             1.01                                                                             31/56                                                                             2.02         0.078                                       13 1073                                                                             5.73 5.59                                                                             5.59                                                                             17/56                                                                             0.12+ HBc    2.594                                       14 1099                                                                             1.72 1.76                                                                             1.94                                                                             10/56                                                                             1.84         0.083                                       15 1118                                                                             5.59 5.79                                                                             5.79                                                                             10/56                                                                             0.31+ HBc    10.5                                        16 1336                                                                             0.93 1.38                                                                             1.38                                                                             18/56                                                                             2.15         0.010                                       17 1501                                                                             5.75 5.67                                                                             5.67                                                                             36/56                                                                             1.99         5.349                                       18 1530                                                                             1.27 1.48                                                                             1.50                                                                             23/56                                                                             2.30         0.943                                       19 1557                                                                             0.91 1.29                                                                             1.28                                                                             20/56                                                                             2.20         0.385                                       20 1652                                                                             2.06 2.64                                                                             2.72                                                                             42/56                                                                             1.72         0.135                                       21 1877                                                                             5.59 5.63                                                                             5.63                                                                             65/56                                                                             2.16  ALT    4.943                                       22 1940                                                                             1.64 1.47                                                                             1.17                                                                             29/56                                                                             2.35         0.052                                       23 2017                                                                             5.60 5.84                                                                             5.84                                                                             11/56                                                                             0.19+ HBc    6.786                                       __________________________________________________________________________

As shown in FIGS. 3-1 and 3-2, the frequency distribution of the HCV-EIAsignal to cutoff ratios, using peptide IIG at 5 ug/mL as the coatingconcentration, that was obtained with 264 normal serum and 264 normalplasma specimens for commercial sources suggested a repeatably reactiverate of 5.3% and 3.4% respectively. These percentages are relativelyhigh compared with those reported in field clinical trials (usually0.5-1.0%) using the rDNA HCV C-100 based EIA kit (Chiron/Ortho).However, in the assay according to the present invention, the mean s/cratios for the negative (n=250) and screened out positive (i.e. n=14)serum specimens are 0.034 and 7.202 respectively; for the negative(n=255) and positive (n=9) normal plasma specimens the mean ratios are0.084 and 7.089 respectively. Such a sharp contrast between the screenedout positives and all the negatives probably precludes the likelihood ofa high false positive rate. Since these normal specimens are derivedfrom commercial plasma centers where the paid donors usually represent apopulation with higher incidence of viral markers than the rigorouslymonitored blood banks, a higher repeatably reactive rate is alsoconsidered reasonable. Previous clinical studies indicated that between7 to 10 percent of patients receiving transfusions developed NANBH,where 90% of these post-transfusion hepatitis cases are caused by theNANBHV(5). These reports also provide some support to the interpretationof the data obtained herein that a high reactivity represents a truepositive result.

Results obtained from the screening of a total of 677 well-characterizedclinical specimens previously categorized into six groups, from (d) to(i) using a representative lot of plates coated with Peptide IIG, wereplotted on a histogram as shown in FIG. 4.

Fifteen out of fifty (i.e. 30%) HBsAg carriers, 3 out of 39 (i.e. 8%)HBc antibody positive individuals, 43 out of 174 (i.e. 24.7%)individuals with elevated ALT enzyme activity, 8 out of 124 (6.5%)asymptomatic individuals with retroviral antibodies, 6 out of 270 (i.e.2.2%) individuals with retroviral related disease, and 0 out of 20 (i.e.0%) individuals with autoimmune disease were found to be repeatablyreactive with the peptide HCV EIA of the present invention using peptideIIG. All these positive specimens were also found to be positive whentested on peptides IIF/IIID HCV EIA, although with much higher s/cratios.

A much higher percentage of positive cases was found with those who haveabnormal liver functions (24.7%) or previous infection(s) with HepatitisB (30% and 8%) when compared to those with other infections or diseases(e.g. 6.5%, 2.2% and 0%).

Note: Sera from HBsAg carriers were kindly provided by the InfectiousDiseases Laboratory of the American Red Cross; sera from HBc antibodypositive donors were obtained from Boston Biomedica Inc.; sera fromindividuals with elevated ALT levels (>100 I.U./L) were obtained fromboth Boston Biomedica Inc. and NABI laboratory; sera from asymptomaticindividuals with retroviral antibodies (HIV-1 and HTLV-1) were obtainedfrom New York Blood Center, and those with HIV-2 antibodies were fromGuinea Bissau of West Africa, kindly provided by Dr. O. Varnier ofItaly,; sera from patients with ATL were kindly provided by the JapaneseRed Cross; sera from patients with AIDS and ARC, were kindly provided byDr. D. Knowles at Columbia University College of Physicians andSurgeons, and Dr. F. Siegal at the Long Island Jewish Hospital; serafrom patients with various complications of autoimmune diseases werekindly provided by Dr. N. Chiorazzi of the Cornell University MedicalSchool. All sera have been characterized by additional licensedserologic markers before inclusion in the current study.

Table 3 illustrates results obtained with the peptide based HCV EIAdescribed in this invention on a panel of 23 recombinant HCV EIArepeatably reactive specimens obtained from a random donor population.Data on each specimen's ALT level and HBc antibody reactivity areprovided as supplemental information for indirect confirmation of NANBHstatus of the positive donors. As can be seen from the Table, all eightspecimens with indirect confirmation of their NANBH status scoredpositive in the peptide based EIA according to the present invention (onboth IIG and IIF/IIID plates). In addition, four specimens that scoredhigh on the peptide based assay also scored as strong positives by therecombinant HCV EIA, thus further confirming the HCV positivity of thesespecimens. Only one specimen scored marginally positive on the peptidebased HCV EIA, which lacks the other markers. However, this specimenscored positively with the recombinant HCV EIA. The remaining tenspecimens that scored negative by the peptide based EIA according to thepresent invention all had a marginal s/cutoff ratio of between 0.9 to2.6. FIG. 5 provides a direct correlation between the peptide based HCVEIA of the present invention and the recombinant based HCV EIA by theirrespective s/cutoff ratios for this panel. Thus, the peptide based HCVEIA of the present invention can clearly differentiate the repeatablyreactive specimens previously screened out by the rDNA based HCV EIAinto two distinct groups, a positive group which correlated highly tothose with other known NANBH markers and a negative group which probablyrepresents specimens with extraneous reactivities unrelated to HCV. Inaddition to its use as a screening assay, the peptide based HCV EIA mayalso function as a positive confirmatory test for the rDNA based HCVEIA.

Note: This well-characterized serum panel was kindly provided by Dr. C.Fang of the American Red Cross QC laboratory.

EXAMPLE 3 Detection of Antibodies to HCV By an Agglutination Based Assay

The presently claimed HCV peptides, synthesized according to theMerrifield solid phase method, can be conjugated to bovine serum albumin(BSA) by a simple crosslinking method in the presence of a lowpercentage of glutaraldehyde solution (0.025%), or with othercrosslinking reagents such as m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS) according to a previously published procedure (Biochemistry,18:690-697, 1979). For example: to 0.32 mL. of a BSA solution (10 mg/mLin 0.01M phosphate buffer, pH 7.0) at room temperature is added 0.013 mLof an MBS solution (0.025 mg/mL in dimethylformamide). The amount of MBSadded to the BSA solution can be varied dependent on the optimal molarratio of BSA to MBS determined for a specific conjugate studied. Themixture is stirred at room temperature for 1 hour, after which it iscentrifuged to remove any precipitated albumin. The clarified mixture isthen subjected to gel filtration on Sephadex G-25 and theprotein-containing fractions, as detected by their absorbance at 280 nm,are pooled and stored frozen at -70° C. until needed.

The peptides are dissolved in H₂ O at 10 mg/mL. A predetermined amountof each peptide solution is added dropwise to the previously activatedBSA-MBS solution and stirred at room temperature for 3 hours. The finalpeptide-BSA conjugates are separated from other free peptides by gelfiltration or extensive dialysis. The ratio of peptide to BSA isdetermined by SDS-PAGE according to conventional methods.

Using the above mentioned peptide-BSA conjugation process, conjugatedpeptide IIG-BSA was absorbed to double aldehyde fixed human 0erythrocytes at pH 4.0. The peptide-conjugate coated erythrocytes werethen treated with NaBH₄ to prevent non-specific protein binding. Thepeptide-conjugate coated erythrocytes were then washed with PBS andincubated with 5% normal human serum-PBS solution. These processed cellswere then used in an agglutination assay for the detection of HCVantibodies in both serum and plasma specimens. The specimens werediluted 1:10 in a sample diluent buffer and an equal volume of theindicator cells (50 uL) was mixed with the diluted specimens. Theagglutination pattern was settled within one hour; and the assay resultswere read by the naked eye and further quantitated by an optical device(manufactured by Olympus Corporation) which gave a P/C ratio, asdetermined by the absorbance readings of the periphery and center of thewells. In this experiment, a P/C ratio of 20 was set as the assay cutoffvalue, i.e. a positive agglutination pattern had a ratio of <20 and anegative pattern, >20.

A total of 20 rDNA HCV EIA repeatably reactive specimens were tested forantibodies to HCV in the above-described HCV passive hemagglutinationassay (PHA) employing Peptide IIG-BSA conjugate as the solid phase. FIG.6 provides a correlation study between the peptide based HCV PHA and therecombinant based HCV EIA by their respective P/C and s/c ratios. Allsamples with s/c EIA ratios higher than 3 were found to be positive withthe HCV PHA test. With the exception of one, all specimens havingborderline s/c ratios (between 0.9 to 2) scored as negative in this PHAtest.

EXAMPLE 4 Detection of Antibodies to HCV By An Agglutination AssayUtilizing As the Solid Phase Immunosorbent Gelatin Particles,Erythrocytes Of Different Animal Species, Or Latex Particles Coated witha Mixture of HCV Peptides

One mL thoroughly washed erythrocytes, gelatin particles, or polystyrenelatex particles are coated with the HCV peptide mixture, or conjugatesthereof at an effective concentration. The peptide mixture, orconjugates thereof, coated cells or particles are then incubated withserially diluted serum samples in the wells of a 96-well U-shapedmicroplate or on a slide. After being left at room temperature for aboutan hour, or a few minutes in the case of latex particle basedmicroagglutination, the settled agglutination pattern on the bottom ofeach well or on the slide is read; and the highest dilution showing apositive reaction is recorded.

This is a one-step assay which can be used for both qualitative andquantitative detection of antibodies to HCV in specimens including seraor biofluids.

EXAMPLE 5

A test kit for detecting HCV antibodies using an agglutination assaycomprises a compartmented enclosure containing multiple microwell platesand other accessory materials for an agglutination assay including (1) abottle of HCV peptide coated erythrocytes, gelatin particles or latexpolystyrene particles; (2) a negative control; and, (3) an inactivatedHCV positive control, and (4) specimen diluent. The procedure describedin Examples 3 and 4 is to be followed.

EXAMPLE 6

An enzyme immunoassay based diagnostic test kit for the detection of HCVantibodies can be constructed. The test kit comprises a compartmentedenclosure containing multiple 96 well plates coated prior to use withthe HCV peptide or peptide mixtures of the present invention in 100 uLpH 9.5 10 mM NaHCO₃ buffer. The kit further comprises materials forenzyme detection in separate sealed containers consisting of: 1) anegative control; 2) an inactivated HCV positive control; 3) specimendiluent; 4) peroxidase labeled-second antibody to human IgG; and 5) acolor change indicator consisting of, for example, orthophenylenediamine(OPD) and hydrogen peroxide in a phosphate citrate buffer. The proceduredescribed in Examples 1 and 2 is to be followed.

In this test kit, 96-well plates, precoated with a peptide or peptidemixture of the present invention, can be replaced by polystyrene beads,or multiple mini-columns filled with controlled pore size glass beads,or nitrocellulose paper strip, precoated with the peptides of thepresent invention for use as the solid phase immunosorbent.

EXAMPLE 7 Immunization with Octameric HCV Peptides for the Elicitationof Sustaining High Titers of HCV Antibodies

In addition to the use of synthetic HCV peptides as immunogens for thegeneration of sequence related anti-HCV antibodies for the ultimatedevelopment of an epitope-based subunit NANBH vaccine, another approachusing a limited sequential propagation of a trifunctional amino acidlysine to form a core that serves as a low-molecular weight matrixcarrier for peptide immunogens can also be applied. The trifunctionalamino acid, Boc-Lys(Boc), is particularly suitable since both N-α andN-ε amino groups are available as reactive ends. Thus, sequentialpropagation of Boc-Lys(Boc) will generate 2^(n) reactive ends. The firstlevel coupling of Boc-Lys(Boc) will produce two reactive amino ends as abivalent carrier. The sequential generations of a second and third stepwith Boc-Lys(Boc) will produce carriers containing four(tetra-valent),and eight (octa-valent) reactive amino ends to which peptide antigensare attached.

The HCV peptides as described in this invention can be incorporated ontothis carrier system as illustrated below for the development ofsustaining high titer HCV antibodies in mammals, including humans.##STR50##

Octameric HCV peptides of the present invention (Table 1) using thesolid phase method of Merrifield are synthesized by an automated peptidesynthesizer, either Applied Biosystems (ABI) Model 430A, or BiosearchModel 9500.

Both acid-labile tert-butyloxycarbonyl (t-Boc) and acid-stable groupsare used for the protection of N-α amino acid and the functional sidechains of the amino acids during the synthesis, respectively. Theoctameric peptides are synthesized by coupling onto a synthetic octamerresin.

An octamer resin is prepared by coupling di-t-Boc Lys onto 0.14 mmol/gMBHA (4-Methyl benzhydrylamine) resin. (Biosearch 9500 is used for thispreparation due to its flexibility in scale). Di-Boc Lys single couplingis followed by two capping reactions (e.g. 0.3M Acetylimidazole in DMFdimethylformamide). The substitution level of synthetic octamer resin isdetermined by Ninhydrin Test.

Duncan Hartly random bred female guinea pigs (two per immunogen),weighing 400-500 gms, are used as the hosts. For initial immunizations,an aliquot of 100 ug octameric HCV Peptide in 0.5 mL PBS is mixed withan equal volume of complete Freund's adjuvant and injected into eachanimal both subcutaneously and intradermally over multiple sites. Aftertwo to three weeks of rest, an identical dosage of the same immunogen isgiven as a boost into each animal except that incomplete Freund'sadjuvant is used. The animals are bled by heart puncture periodically tomonitor each serum's anti-HCV titers. Subsequent booster shots are givenperiodically.

EXAMPLE 8 Relative (%) Immunoreactivity for Synthetic Peptides By AnEnzyme-Linked Immunoadsorbent Assay

Wells of 96-well plates were coated at 4° C. overnight (or 1 hour at 37°C.), with each of the additional nine peptides, VA, VB, VC, VD, VE (=V),VIA, VIB, VIC, VID, VIE (see Table 1 for the above mentioned peptides),at 5 ug/mL at 100 uL per well in 10 mM NaHCO₃ buffer, pH 9.5. Eachpeptide's immunoreactivity was measured as previously described inExample 1. Results obtained for the 10 peptides in the V and VI seriesare shown in Table 2. According to the EIA absorbance readings at 492nm(y axis) and the amino acid sequences for each of the corresponding HCVpeptides (x axis), representative immunoreactivity profiles are plottedfor four of the eight sera on the 10 peptides in the V and VI series,together with the first twenty peptides in the I, II and III series, asshown in FIGS. 1-1 to 1-4. Relative (%) immunoreactivity index for eachof the additional 10 peptides is likewise calculated using peptide IIIDas a reference. Additional clusters of residues, such as ASRQA andEVIAP, that are identified with these 10 peptides, were found tocontribute additionally to the overall HCV antibody reactivity.

In summary, epitope mapping analysis conducted with a series ofoverlapping peptides reveals a varying degree of immunoreactivitiesbetween different HCV antibody positive samples and these HCV peptides.Based on the above-mentioned epitope mapping study, a thirdrepresentative EIA using peptides IIF, IIID and V as the solid phaseantigen was also configurated for testing as shown in Example 9 incomparison to that using peptide IIF and IIID.

EXAMPLE 9 Detection of Antibodies to HCV in Serial Samples byEnzyme-Linked Immunosorbent Assays

(a) A coded panel consisting of 24 samples derived from a case oftransfusion transmitted NANBH were tested in two types of EIAs usingplates coated with either a mixture of IIF and IIID at 5, 5 ug/mL or amixture of IIF, IIID and V at 5,5,5 ug/mL. The panel was provided by Dr.H. Alter of NIH and the results were decoded by his laboratory.

As shown in FIG. 7-1, the two anti-HCV profiles, as tested by twoformats, using Peptides IIF/IIID/V coated plate (Curve A) and PeptidesIIF/IIID coated plate (curve B) respectively, spanning a ten year periodrevealed an interesting contrast.

According to the record, the seronegative patient received HCVcontaminated blood units on Aug. 20, 1980. As a result of thetransfusion, a trace amount of passive HCV antibodies was detected inthe recipient's serum by format A. Active development of HCV antibodiesby the recipient became detectable by both formats from November 14th on(about three months after the initial transfusion). The HCV antibodies,developed as a result of HCV infection through blood transfusion,persisted throughout the next ten years. Higher antibody signals weredetected by plates coated with an extra peptide V (curve A) in seracollected four months after the transfusion. It appears that the epitopepresented by peptide V, representing a neighboring immunodominantregion, elicits abundant HCV antibodies at a slightly later stage thanthe epitopes represented by peptides IIF and IIID.

(b) Serial samples from one representative case of a hemodialysispatient with NANBH were provided by Dr. Cladd Stevens of New York BloodCenter, N.Y., N.Y., and tested on plates coated with a mixture of threepeptides, IIIF/IID/V. The sample histories are shown in FIG. 7-2. Theresults show that the peptide based EIA detects samples about two monthsafter the onset of the acute phase of the disease as evidenced by theALT elevation.

(c) Serial samples from a representative chimpanzee were tested with apeptide based HCV EIA using a mixture of IIIF/IID/V peptides. Thischimpanzee was inoculated on day 0 with a well-characterized strain ofNANBHV. Following the acute phase of infection as evidenced by the riseof the ALT levels, antibodies to HCV were detected about 60 days afterinoculation [FIG. 7-3].

EXAMPLE 10 Screening of Low Risk Random Blood Donors With the PeptideBased HCV EIA

2035 donor specimens obtained in a blood bank setting were tested by EIAcoated with a mixture of Peptides IIF, IIID and V at 5 ug/mL eachfollowing the procedure described in Example 2. The results are shown inFIGS. 8-1 and 8-2. The frequency distribution of the peptide basedHCV-EIA signal to cutoff ratios, suggested an initial reactive rate of1.18% and a repeatably reactive rate of 1.08 respectively. 88% of theinitial reactive specimens are repeatably reactive indicating a highreproducibility of the assay. The repeatably reactive rate of thepeptide based HCV EIA obtained with the low risk random blood donorspecimens, all volunteers, is lower than that obtained from thecommercial paid donor population (See Example 2).

EXAMPLE 11 Synthetic Peptide Based HCV Neutralization EIA As aConfirmatory Test

Wells of 96-well plates were coated at 4° C. overnight (or for 1 hour at37° C.) with a mixture of two peptides IIF and IIID at 5 ug/mL each in100 uL 10 mM NaHCO₃ buffer pH 9.5. Repeatably reactive specimenspreviously screened out by the direct HCV EIA were incubated with eithera control specimen diluent buffer (i.e., PBS containing 20% by volumenormal goat serum, 1% by weight gelatin and 0.05% by volume Tween 20) ata dilution of 1:20 volume to volume, or with the same specimen diluentbuffer containing varying amounts of a HCV peptide analogue IV (seeTable 1 for its amino acid sequence) and allowed to react for an hour at37°.

200 uL of the peptide IV neutralized specimens were then added to eachof the wells and allowed to react for 15 minutes at 37°, followed by theEIA procedure as described in Example 2. Four representative reactivesamples including two weakly reactives and two strongly reactives weretested. One of the strong reactives was further diluted at 1:10 in thespecimen diluent prior to neutralization testing. As shown in FIG. 9 andTable 4, a dose dependent inhibition [or neutralization] of HCV EIA wasobserved with peptide Iv. When compared with the controls, a significantinhibition was obtained with all four specimens even at a concentrationof 50 ug/mL peptide IV.

EXAMPLE 12 Detection of Antibodies to HCV in Hemodialysis patients byEIA

A coded panel consisting of 74 samples from a group of hemodialysispatients was tested in two types of EIAs using plates coated with amixture of HCV peptides IIH and V at 10,5 ug/mL or a recombinant HCVprotein based EIA. The panel was provided by investigators at theJapanese National Institute of Health and the results were decoded andcompared to the recombinant HCV protein based EIA by the sera provider.

As shown in FIG. 10, an x-y plot of the A492 nm readings for the peptidebased HCV EIA and the recombinant HCV protein based HCV EIA revealed ahigh correlation between these two assays. (A cutoff value of 0.2 and0.4 was obtained based on the corresponding assay design.) These 74specimens obtained from the hemodialysis patients who are highlysusceptible to HCV infection were grouped into four categories based ontheir respective reactivities with these two types of EIAs. The upperright block indicates samples that are scored positive by both assays,and the lower left block indicates samples that are scored negative byboth assays. None of the 74 high risk samples were found positive by therecombinant based EIA and negative by the peptide based EIA as shown inthe upper left block; whereas five of these 74 high risk samples scoredpositive by the peptide based EIA and negative by the recombinant basedEIA as shown in the lower right block indicating that the peptide basedHCV EIA is more sensitive when tested with specimens derived frompatients at high risks for HCV infection.

EXAMPLE 13 Detection Of Anti-HCV Activity In Rare Specimens With AnElevated ALT Level

These results are representative of the acute phase of HCV infection bysynthetic peptides of Peptide VII series, covering a region near theC-terminus of the HCV protein C-100. and Peptide IIH from theimmunodominant region.

Wells or 96-well plates were coated at 37° C. for 1 hour with each ofthe six peptides VIIA, VIIB, VIIC, VIID, IIG and IIH, at 5 ug/mL at 100uL per well in 10 mM NaHCO₃ buffer, pH 9.5. Each peptide'simmunoreactivity with the respective specimen was measured as previouslydescribed in Example 1. As shown in Table 5, weak immunoreactivity wasobtained with specimen 3 for peptides VIIC and VIID, but not VIIA andVIIB. Moderate immunoreactivity was obtained with specimen NAB-2-2 forpeptide IIH, but not IIG. Both specimens were found to have high ALTlevel and are representative of specimens from patients with acute phaseof HCV infection.

                                      TABLE 4                                     __________________________________________________________________________    Peptide Based HCV Neutralization EIA                                          As A Confirmatory Test                                                                Peptide IV Concentration                                              Specimens                                                                             400 ug/mL                                                                            200 ug/mL                                                                             100 ug/mL                                                                             50 ug/mL                                                                              Control                                ID Dilution                                                                           mA % I mA  % I mA  % I mA  % I mA                                     __________________________________________________________________________    A  1:1  590                                                                              72.0                                                                              1129                                                                              46.5                                                                              1066                                                                              49.5                                                                              1363                                                                              35.4                                                                              2111                                       1:10                                                                              244                                                                              86.1                                                                              325 81.5                                                                              409 76.7                                                                              510 71.0                                                                              1762                                   B  1:1  161                                                                              92.0                                                                              209 89.6                                                                              321 84.1                                                                              523 74.1                                                                              2021                                   C  1:1  117                                                                              89.0                                                                              162 84.7                                                                              155 85.4                                                                              153 85.6                                                                              1064                                   D  1:1   27                                                                              89.8                                                                               23 91.3                                                                               38 85.7                                                                               34 87.2                                                                               266                                   __________________________________________________________________________     ##STR51##                                                                

                                      TABLE 5                                     __________________________________________________________________________                                                     A492 nm                                                                       Specimen No.                 Code                                                                              Amino Acid Sequence                          NAB-2-2                                                                            #3                      __________________________________________________________________________    IIH SG, KPAII, PDREV, LYREF, DEMEE, CSQHL, PYIEQ, GMMLA,                                                                  KALGL,                                                                             1.232(+)                     IIG II, PDREV, LYREF, DEMEE, CSQHL, PYIEQ, GMMLA, EQFKQ,                                                                  KALGL                                                                              0.013(-)                     VIIA                                                                              AVQWM, NRLIA, FASRG, NHVSP                        0.109                   VIIB                                                                              RHV, GPGEG, AVQWM, NRLIA, FASRG, NHVSP            0.224                   VIIC                                                                              V, VCAAI, LRRHV, GPGEG, AVQWM, NRLIA, FASRG, NHVSP                                                                              0.674                   VIID                                                                              PGA, LVVGV, VCAAI, LRRHV, GPGEG, AVQWM, NRLIA, FASRG,                                                                           0.658                   __________________________________________________________________________

EXAMPLE 14 Measurement Of Relative (%) Immunoreactivity For SyntheticPeptide Covering An Immunodominant Region Of The Postulated HCV CoreProtein By An Enzyme-Linked Immunosorbent Assay

Wells of 96-well plates were coated at 4° C. overnight (or 1 hour at 37°C.), with each of the ten peptides: VIIIA, VIIIIB, VIIIC, VIIID,VIIIE(=VIII), IXA, IXB, IXC, IXD, and IXE(=IX), (see Table 7) preparedas described at 5 ug/mL at 100 uL per well in 10 mL NaHCO3 buffer, pH9.5. The rest of the plate coating and enzyme immunoassay procedureswere performed exactly as described in Example 1.

Results obtained from this study are shown in FIGS. 11-1 and 11-2.According to the EIA absorbance readings at 492 nm (y axis) and theamino acid sequences for each of the corresponding HCV peptides (xaxis), representative immunoreactivity profiles are plotted for four ofthe eight panel sera as shown in FIGS. 11-1 and 11-2. Relative (%)immunoreactivity index for each of the 10 peptides (Table 7) iscalculated against Peptide IIID (See Table 1), the one with the highestabsorbance reading, based on the total absorbance of eight sera at 492nm (See Tables 1 and 2 for examples of calculation) FIGS. 11-1 and 11-2show the amino acid sequences of the immunodominant region according todata obtained for immunoreactivity study. For example, serum sample 1reacted strongly (ODs between 1.5 and 3.5) with peptides VIIIA and IXA,which are the smallest size in the 20 mer range in the correspondingpeptide series Further addition of amino acids at the N-terminal end didnot significantly enhance the immunoreactivity of these analogue peptide(see FIGS. 11-1 and 11-2 for sample 1).

However, other serum samples such as #3 and #4 reacted much strongerwith peptides VIIIB, VIIIC respectively and with an increasingimmunoreactivity with the analogue peptides in the IX series (see FIGS.11-1 and 11-2, samples #3 and #4). Further, serum sample 2 reactedmarginally with peptides in the corresponding VIII and IX series. Thesereactivity profiles indicate a more complicated epitope distributionalong the postulated HCV core protein region and may include somediscontinuous linear epitopes and conformational epitopes, requiring alonger size peptide to confer the best immunoreactivity for diagnosticpurposes.

In summary, epitope mapping analysis conducted with a series of tenpeptides covering an immunodominant region of the postulated HCV coreprotein, spanning a total of 119 amino acid residues as illustrated inTable 7 and FIGS. 11-1 and 11-2, reveals varying degrees ofimmunoreactivity between different HCV antibody positive serum samplesand analogue HCV Peptides of the VIII and IX series. In this case it isfound preferably to have synthetic peptides with longer amino acidchains, ideally longer than 20, to optimally present these antigenicdeterminants to HCV antibodies.

Based on the above-mentioned epitope mapping study, additionalrepresentative EIAs using Peptides VIIIE, IXD, both derived from the HCVcore region alone, or as a mixture with peptides IIH and V from the HCVnonstructural region were configurated for the following studiesdescribed in Examples 15, 16, 17 and 18.

EXAMPLE 15 Detection Of Antibodies To HCV By Peptide Based Enzyme-LinkedImmunosorbent Assay Using Format C. Format D. Format A

The following four groups of specimens:

(a) individuals with AIDS, ARC(n=63);

(b) individuals positive for HBsAg, (n=50);

(c) individuals positive for antibodies to HBc protein, (n=22); and

(d) individuals with elevated (>100 i.u./L) alanine aminotransferase(ALT) enzyme activity, (n=86).

were analyzed on representative HCV peptide based EIAs according to thepresent invention, with the plates coated either with (i) peptides IIHand V at 5 and 3 ug/mL each (Format A), (ii) peptides IIH, V and VIIIEat 5, 3 and 2 ug/mL each (Format C, containing both the HCV core andnonstructural peptides) or (iii) Peptides VIIIE and IXD at 2 and 2 ug/mLeach (Format D, HCV core peptides only).

Results obtained from the screening of a total of 221 well-characterizedclinical specimens previously categorized into four groups, from (a) to(d) using a representative lot of peptide coated plates EIAs formattedas A, C or D were plotted on histograms as shown in FIGS. 12-1, 12-2 and12-3.

Out of a total of 63 AIDS/ARC patient samples analyzed, 46.0%, 55.6% and50.8% of the patients were found to be HCV antibodies positive using EIAformats A, C and D respectively. Out of 50 HBsAg positive individuals,36.0%, 42.0% and 36% of the individuals were found to also be HCVantibodies positive using EIA formats A, C and D respectively. Out of 22HBc antibody positive individuals, 27.3%, 22.7%, and 18.2% were found tobe HCV antibodies positive as detected by EIA formats A, C and D. Out of86 patients with an elevated ALT levels, 90.7%, 91.5% and 85.4% werefound to be HCV antibodies positive by EIA formats A, C and D. Theoverall signal to noise ratio distribution for the HCV positive sampleswere found to be higher with Formats C and D which included a peptide(VIIIE) from the HCV core region than Format A which only employedpeptides from the HCV nonstructural region as the solid phase antigen.

Except for one HBc antibody sample where the results is borderlinepositive (S/cutoff ratio˜1.0) with the HCV EIA Format A, Format Cincorporating peptides (IIH, V and VIIIE) from both the HCV structural(core) and nonstructural regions was the most sensitive. The significantimprovement in sensitivity makes Format C an ideal candidate for a HCVantibody screening assay.

EXAMPLE 16 Comparison Of Test Results Using The Three Peptide Based HCVEIA Formats (A,C And D) On Low Risk Random Blood Donors

Representative 264 donor specimens obtained in a blood bank setting weretested by all three EIA formats.

The results are shown in FIGS. 13-1 to 13-6. The frequency distributionsof the peptide based HCV-EIA signal to cutoff ratios suggested aninitial reactive rate of 1.13%, 3.0% and 3.0% with formats A, C and Drespectively. The negative samples have a relative low signal to cutoffratio in all three assay formats (see FIGS. 13-1, 13-3, and 13-5). Uponrepeat testing, a repeatably reactive rate of 1.13%, 1.9% and 1.9% wereobtained for formats A, C and D respectively. Among the sera identifiedas positives, there were four specimens which reacted strongly with bothFormats C and D, but were identified as negatives by Format A. Thisindicates the possibility of false negative results when an HCV antibodydetection assay does not include epitopes from the structural proteinregion.

EXAMPLE 17 Detection Of Antibodies To HCV In Well-Characterized SerialSamples By Various Enzyme-Linked Immunosorbent Assays

(a) A coded panel consisting of 24 samples derived from a case oftransfusion transmitted NANBH were tested in three HCV EIA formats (A, Band C) to determine the respective sensitivity of the formats indetecting seroconversion. The panel was provided by Dr. H. Alter of NIHand the results were decoded by his laboratory.

As shown in FIG. 14-1, the three anti-HCV profiles, as tested by threeformats using peptides IIF/IIID/V coated plate (Curve A); PeptidesIIF/IIID coated plate (Curve B); and peptides IIH, v and VIIIE coatedplate (Curve C) respectively, with sera spanning a ten year periodrevealed an interesting contrast.

As a result of the transfusion, a trace amount of passive HCV antibodieswas detected in the recipient's serum by both format A, and C. Activedevelopment of HCV antibodies by the recipient became detectable by allthree formats from November 14th on (about three months after theinitial transfusion), with format C having the highest S/cutoff ratio onthat bleed date. This finding further confirms the improved sensitivityobtained by using HCV EIA format C.

(b) Serial samples from a representative chimpanzee were tested with HCVEIA format C in comparison to a recombinant HCV C-100 basedradioimmunassay (RIA). The chimpanzee was inoculated on day 0 with awell-characterized strain of NANBHV. Following the acute phase ofinfection as evidenced by the rise of the ALT levels, antibodies to HCVwere detected about 47 days after inoculation (FIG. 14-2). HCV EIAFormat C was able to detect HCV antibodies about 40 days early than therDNA based RIA. A higher signal to cutoff ratio was obtained with HCVEIA Format C than the rDNA RIA.

(c) Serial samples from three well-characterized representative HCVseroconversion panels, collected by Serologic Inc., were tested by HCVEIA formats A, C and D, as defined in Example 15 in addition to thatpreviously tested with rDNA HCV C-100 based EIA. As shown in Table 8,both HCV EIA formats C and D were able to identify HCV antibody positivespecimens in two out of three panels by four to eight weeks earlier thanthe rDNA HCV-100 based EIA and HCV EIA Format A. This furtherdemonstrated the sensitivity of the HCV EIAs which incorporate peptidesderived from the HCV structural (core) protein region.

EXAMPLE 18 Detection Of Antibodies To HCV In Hemodialysis Patients ByVarious Forms Of HCV EIAs

A coded panel consisting of 74 samples from a group of hemodialysispatients was tested by three types of HCV EIAs; a recombinant HCVprotein based EIA, and two using plates coated with either a mixture ofHCV peptides IIH and V at 10, 5 ug/mL respectively (Format A), or amixture of HCV peptides IIH, V and VIIIE at 5, 3 and 2 ug/mLrespectively (Format C). The panel was provided by investigators at theJapanese National Institute of Health. Results were decoded and comparedto the recombinant HCV protein based EIA by the sera provider.

As shown in FIG. 15-1, an x-y plot of the A492 nm readings for thepeptide based HCV EIA Format C and the recombinant HCV protein based HCVEIA revealed an increased sensitivity with the peptide based HCV EIAformat C when compared to the rDNA HCV C-100 protein based HCV EIA. (Acutoff value of 0.2 and 0.4 was obtained based on the correspondingassay design). These 74 specimens obtained from dialysis patients whoare highly susceptible to HCV infection were grouped into fourcategories based on their respective reactivities with these two typesof EIAs. The upper right block indicates samples that were scoredpositive by both assays, and the lower left block indicates samples thatwere scored negative by both assays. None of the 74 high risk sampleswere found positive by the recombinant based EIA and negative by thepeptide based EIA as shown in the upper left block; whereas "eleven" ofthese 74 high risk samples scored positive by the peptide based EIAFormat C and negative by the recombinant based EIA as shown in the lowerright block.

An increase in sensitivity was obtained for the peptide based HCV EIAFormat C (incorporating a HCV core peptide) when compared to HCV EIAFormat A, which in turn showed an improved sensitivity compared with therecombinant HCV C-100 protein based EIA (see Example 12, FIG. 10).

To further document the validity of such a sensitivity comparison, otherclinical data obtained for each of the dialysis patient specimens weretabulated along with the corresponding EIA ratios (Table 9). Among theeleven marked specimens, most showed an increased level of GOT/GPT andwere associated with frequent episodes of elevated GPT previously. Alleleven specimens scored negative by the rDNA HCV C-100 based EIA.However, these same samples reacted strongly (with O.D.˜1.5) in thepeptide based HCV EIA Format C. Since peptide VIII(=VIIIE) wassynthesized according to amino acid sequences selected from theconserved structural (core) protein region, its inclusion in the peptidebased HCV EIA (such as format C) will be particularly suitable whentesting specimens from geographically distinct regions where a higherchance of strain-to-strain variation among the HCV isolates may beencountered.

It is to be understood that the above examples are illustrative of thepresent invention and are not meant to limit the scope thereof.

                                      TABLE 8                                     __________________________________________________________________________    Testing of Various Formats of HCV EIAs with Three Well-Characterized          Seroconversion Panels                                                                                EIA Ratio                                                                            HCV EIA                                                                             HCV EIA                                                                             HCV EIA                                 Donor     ALT  AST rDNA HCV                                                                             Format A                                                                            Format C                                                                            Format D                            Panel                                                                             #   Bleed Date                                                                          I.U./L                                                                             I.U./L                                                                            c-100  (ns)  (core + ns)                                                                         (core)                              __________________________________________________________________________    Panel 1                                                                           02190D                                                                            880809                                                                              40.0 NA  0.03   0.093 0.108 0.205                                       880816                                                                              32.0 NA  0.04   -0.014                                                                              0.045 0.129                                       880823                                                                              32.0 NA  0.06   -0.050                                                                              0.025 0.072                                        880830*                                                                            180.0                                                                              121.0                                                                             0.04   -0.050                                                                              1.037*                                                                              1.096*                                      880928                                                                              401.0                                                                              352.0                                                                             0.19   0.100 7.193 7.703                                        881109*                                                                            NA   NA  6.57*  16.700*                                                                             10.185                                                                              7.281                                       881122                                                                              NA   NA  6.57   16.671                                                                              9.770 9.321                               Panel 2                                                                           00269B                                                                            880815                                                                              39.0 NA  0.0    0.014 -0.058                                                                              -0.008                                      880825                                                                              274.0                                                                              310.0                                                                             0.0    0.443 0.058 0.108                                       880829                                                                              346.0                                                                              270.0                                                                             0.0    0.029 0.128 0.185                                       880914                                                                              1175.0                                                                             722.7                                                                             6.5*   4.057 7.835*                                                                              5.984*                                      881005                                                                              429.7                                                                              172.3                                                                             6.5    5.857 7.811 5.851                               Panel 3                                                                           20830D                                                                            880829                                                                              63.0 65.0                                                                              0.04   -0.043                                                                              0.115 0.181                                        880901*                                                                            81.0 NA  0.04   0.043 1.607*                                                                              1.108*                                      880908                                                                              183.0                                                                              174.0                                                                             0.02   -0.043                                                                              2.506 3.116                                        880928*                                                                            563.0                                                                              555.0                                                                             6.57*  3.800 9.827 9.659                                       881026                                                                              436.0                                                                              151.0                                                                             6.57   13.786                                                                              10.630                                                                              10.566                              __________________________________________________________________________

                                      TABLE 9                                     __________________________________________________________________________    HCV Positivity in Serum Specimens                                             Obtained from Japanese Dialysis Patients                                           rDNA  Peptide                                                                             Peptide                                                           based based based            n: times                                         HCA   HCV EIA                                                                             HCV EIA          during                                           EIA OD                                                                              Format A                                                                            Format C         1986-1988                                        Cutoff =                                                                            Cutoff =                                                                            Cutoff =   GOT/GPT                                                                             when GPT                                    Code No.                                                                           0.40  0.205 0.204 HBsAb                                                                              Oct., 89                                                                            >25 I.U./L                                  __________________________________________________________________________    24   0.058 -0.001                                                                              0.005      2/3   0                                           25   0.042 0.005 0.007      9/9   0                                           26   0.105 -0.001                                                                              -0.003     4/4   0                                           27   1.837 1.469 2.312 -    3/6   2                                           28   1.797 1.637 2.398 -    20/21 2                                            29* 0.011 0.001 1.603      7/4   0                                           30   0.994 0.374 2.213      11/9  0                                           31   1.823 0.425 0.874 -    27/16 4                                           32   0.770 0.372 0.500 +    17/7  9                                           33   1.712 2.101 2.234 -    28/32 29                                          34   0.002 -0.003                                                                              0.007      11/14 0                                            35* 0.026 0.161 2.229 +    14/23 23                                           36* 0.065 0.018 2.286      20/18                                             37   0.021 0.000 0.011 +    16/11 1                                           38   2.347 1.917 2.182 +    26/23 6                                           39   0.008 -0.007                                                                              0.004      7/6   0                                           40   0.026 0.006 -0.002     10/8  0                                            41* 0.061 0.118 1.933 +    9/6                                               42   2.481 2.144 2.211 -    13/19 2                                           43   0.008 -0.005                                                                              -0.005                                                                              +    11/7                                              44   0.009 -0.004                                                                              -0.005     4/4   0                                           45   0.009 0.000 -0.003     7/2   0                                           46   2.177 1.990 2.121 -    16/12 8                                           47   0.023 0.003 0.015      7/3   0                                           48   0.025 -0.003                                                                              0.002 +    18/11                                             49   0.025 -0.001                                                                              -0.006     9/5   0                                           50   0.026 0.024 -0.003     9/3                                               51   0.018 -0.003                                                                              -0.007                                                                              +    11/5                                               52* 0.011 -0.003                                                                              1.366 -    33/52 29                                          53   2.251 1.296 2.218      8/7   0                                           54   0.050 0.017 0.040      10/7  0                                           55   0.020 -0.007                                                                              0.017 +    14/8                                              56   0.033 -0.004                                                                              0.000      9/3   0                                           57   1.396 0.718 2.121 -    17/11 1                                           58   0.045 0.013 -0.003     13/12                                             59   0.014 0.068 0.056      10/7  0                                           60   0.009 0.014 0.056 +    15/0  10                                          61   2.007 2.214 2.235 +    12/9                                              62   0.171 0.001 0.003      11/7  0                                           63   1.121 0.529 2.383 +    18/10                                             64   0.113 0.066 0.002      4/3   0                                           65   0.032 0.003 -0.003                                                                              +    7/5   3                                           66   0.039 -0.001                                                                              -0.002                                                                              +    11/6                                               67* 0.049 0.037 2.119      16/11                                              68* 0.177 0.638 2.000 +    24/25 33                                          69   0.027 0.007 -0.007     6/3   0                                           70   0.031 -0.006                                                                              -0.001     16/9  0                                           71   0.781 0.473 2.151 +    13/8  14                                          72   0.110 0.002 0.059      13/8  0                                           73   0.043 -0.002                                                                              -0.007                                                                              -    2/3   0                                           74   0.014 0.001 -0.004     2/3   0                                           75   0.053 0.000 0.019 +    15/8                                              76   0.060 0.015 0.018      14/7  0                                           77   0.011 0.001 -0.004     8/8                                               78   0.042 0.002 0.023      3/0   0                                           79   0.537 0.219 1.742 +    11/7                                              80   2.615 1.713 2.428 +    18/16 12                                          81   2.509 2.265 2.294      9/4                                               82   0.019 0.000 0.120      11/5  0                                           83   0.511 1.928 2.229 -    19/11 5                                           84   0.020 0.016 0.095 -    12/9                                              85   0.013 -0.003                                                                              0.116      10/7  0                                           86   0.003 -0.005                                                                              -0.006     19/5                                              87   0.031 -0.009                                                                              0.009      10/6  0                                           88   0.039 0.019 0.004      6/2   0                                            89* 0.273 0.223 2.055 -    10/8  8                                           90   0.045 0.026 -0.002                                                                              -    7/3   3                                           91   0.018 0.003 -0.002     5/8   0                                           92   1.974 1.127 2.189 +    11/23 22                                          93   0.893 1.113 2.226 -    24/19 5                                            94* 0.267 0.353 2.029 -    18/12 1                                           95   0.026 -0.010                                                                              0.000 -    34/73 0                                            96* 0.021 0.002 1.599 -    13/30 27                                           97* 0.246 0.037 1.779      15/9  0                                           98   2.412 1.904 2.236 -    3/9                                               __________________________________________________________________________

We claim:
 1. A peptide composition comprising a peptide selected fromthe group consisting of Peptide I, III to IX, each peptide with an aminoacid sequence as follows: ##STR52## wherein X is --OH or --NH₂ ; and(ix) a. an analogue of each of the above peptides having an amino acidsequence derived from a strain/isolate of HCV in a region correspondingto the peptide and having specific immunoreactivity to antibodies to HCVrelative to the peptide that is substantially preserved;b. a segment ofeach of the above peptides or analogue thereof having specificimmunoreactivity to antibodies to HCV relative to the peptide of atleast 17.8% up to 101%; c. a mixture of the above peptides or analoguesof the peptides; d. a conjugate of each of the peptides with carrierproteins, the conjugate having specific immunoreactivity to antibodiesto HCV relative to the peptide of at least 17.8% up to 101%; and e. apolymer of each of the peptides comprising a branching dimer, tetramer,or octomer of the peptide on a mono, tri, or hepta lysine corerespectively.
 2. A peptide composition comprising Peptide II having anamino acid sequence as follows: ##STR53## and an analogue thereof havingan amino acid sequence derived from a strain/isolate of HCV in a regioncorresponding to said peptide and having specific immunoreactivity toHCV relative to said peptide that is substantially preserved.
 3. Apeptide composition according to claim 2 wherein the peptide comprises asegment of Peptide II and has an amino acid sequence selected from thegroup consisting of: ##STR54## wherein X is --OH or --NH₂ and ananalogue of each of the above peptides having an amino acid sequencederived from a strain/isolate of HCV in a region corresponding to thepeptide and having specific immunoreactivity to antibodies to HCVrelative to the peptide that is substantially preserved.
 4. A peptidecomposition according to claim 1 wherein the peptide comprises a segmentof Peptide III and has an amino acid sequence selected from the groupconsisting of: ##STR55## wherein X is --OH or --NH₂ and an analogue ofeach of the above peptides having an amino acid sequence derived from astrain/isolate of HCV in a region corresponding to the peptide andhaving specific immunoreactivity to antibodies to HCV relative to thepeptide that is substantially preserved.
 5. A peptide compositionaccording to claim 4 wherein the peptide has an amino acid sequence asfollows: ##STR56## wherein X is --OH or --NH₂ or an analogue of theabove peptide having an amino acid sequence derived from astrain/isolate of HCV in a region corresponding to the peptide andhaving specific immunoreactivity to antibodies to HCV relative to thepeptide that is substantially preserved.
 6. A peptide compositionaccording to claim 1 wherein the peptide comprises a segment of PeptideVIII and has an amino acid sequence selected from the group consistingof: ##STR57## wherein X is --OH or --NH₂ or an analogue of each of theabove peptides having an amino acid sequence derived from astrain/isolate of HCV in a region corresponding to the peptide andhaving specific immunoreactivity to antibodies to HCV relative to thepeptide that is substantially preserved.
 7. A peptide compositionaccording to claim 1 wherein the peptide comprising a segment of PeptideIX and has an amino acid sequence selected from the group consisting of:##STR58## wherein X is --OH or --NH₂ or an analogue of each of the abovepeptides having an amino acid sequence derived from a strain/isolate ofHCV in a region corresponding to the peptide and having specificimmunoreactivity to antibodies to HCV relative to the peptide that issubstantially preserved.
 8. A peptide composition according to claim 1wherein the peptide has an amino acid sequence as follows: ##STR59##wherein X is --OH or --NH₂, or an analogue of the above peptide havingan amino acid sequence derived from a strain/isolate of HCV in a regioncorresponding to the peptide, and having specific immunoreactivity toantibodies to HCV relative to the peptide that is substantiallypreserved; and a segment of the above peptide having specificimmunoreactivity to antibodies to HCV relative to the peptide of atleast 17.8% up to 101%.
 9. A peptide composition according to claim 1wherein the peptide has an amino acid sequence as follows: ##STR60##wherein X is --OH or --NH₂, an analogue of the above peptide having anamino acid sequence derived from a strain/isolate of HCV in a regioncorresponding to the peptide, and having specific immunoreactivity toantibodies to HCV relative to the peptide that is substantiallypreserved; and a segment of the above peptide or analogue thereof havingspecific immunoreactivity to antibodies to HCV relative to the peptideof at least 17.8% up to 101%.
 10. An enzyme linked immunosorbent assay(ELISA) test kit for the detection of antibodies to HCV or NANBHV or thediagnosis of HCV or NANBHV infection comprising:(i) a solid substratecoated with a peptide composition according to claim 1; (ii) a negativecontrol sample; (iii) an inactivated HCV positive control sample; (iv)specimen diluent; (v) enzyme labelled antibodies to human IgG; and (vi)an enzyme substrate.
 11. An ELISA test kit according to claim 28 whereinthe solid phase is coated with a peptide composition comprising asegment of Peptide II and having an amino acid sequence selected fromthe group consisting of: ##STR61## wherein X is --OH or --NH₂, and ananalogue of each of the above peptides having an amino acid sequencederived from a strain/isolate of HCV in a region corresponding to thepeptide and having specific immunoreactivity to antibodies to HCVrelative to the peptide that is substantially preserved.
 12. An ELISAtest kit according to claim 10 wherein solid phase is coated with apeptide having an amino acid sequence: ##STR62## wherein X is OH or--NH₂ and an analogue of the above peptide having an amino acid sequencederived from a strain/isolate of HCV in a region corresponding to thepeptide and having specific immunoreactivity to antibodies to HCVrelative to the peptide that is substantially preserved.
 13. An ELISAtest kit according to claim 10 wherein the solid phase is coated with apeptide composition comprising a segment of Peptide III having an aminoacid sequence selected from the group consisting of: ##STR63## wherein Xis --OH or --NH₂ and an analogue of each of the above peptide having anamino acid sequence derived from a strain/isolate of HCV in a regioncorresponding to the peptide and having specific immunoreactivity toantibodies to HCV relative to the peptide that is substantiallypreserved.
 14. An ELISA test kit according to claim 13 wherein thepeptide is: ##STR64## wherein X is --OH or --NH₂ or an analogue of theabove peptides having an amino acid sequence derived from astrain/isolate of HCV in a region corresponding to the peptide andhaving specific immunoreactivity to antibodies to HCV relative to thepeptide that is substantially preserved.
 15. An ELISA test kit accordingto claim 10 wherein solid phase is coated with a peptide compositioncomprising a segment of Peptide VIII having an amino acid sequenceselected from the group consisting of: ##STR65## wherein X is --OH or--NH₂ or an analogue of each of the above peptides having an amino acidsequence derived from a strain/isolate of HCV in a region correspondingto the peptide and having specific immunoreactivity to antibodies to HCVrelative to the peptide that is substantially preserved.
 16. An ELISAtest kit according to claim 10 wherein solid phase is coated with apeptide composition comprising a segment of Peptide IX having an aminoacid sequence selected from the group consisting of: ##STR66## wherein Xis --OH or --NH₂ or an analogue of each of the above peptides having anamino acid sequence derived from a strain/isolate of HCV in a regioncorresponding to the peptide and having specific immunoreactivity toantibodies to HCV relative to the peptide that is substantiallypreserved.
 17. An ELISA test kit according to claim 10 wherein the solidphase is coated with a peptide composition comprising a peptide havingan amino acid sequence as follows: ##STR67## wherein X is --OH or --NH₂or an analogue of the above peptide having an amino acid sequencederived from a strain/isolate of HCV in a region corresponding to thepeptide, and having specific immunoreactivity to antibodies to HCVrelative to the peptide that is substantially preserved; and a segmentof the above peptide or analogue thereof having specificimmunoreactivity to antibodies to HCV relative to the peptide of atleast 17.8% up to 101%.
 18. An ELISA test kit according to claim 10wherein the solid phase is coated with a peptide composition comprisinga peptide having an amino acid sequence as follows: ##STR68## wherein Xis --OH or --NH₂ and an analogue of each of the above peptides having anamino acid sequence derived from a strain/isolate of HCV in a regioncorresponding to the peptide and having specific immunoreactivity toantibodies to HCV relative to the peptide that is substantiallypreserved.
 19. An ELISA test kit according to claim 10 wherein the solidphase is coated with a peptide composition comprising a mixture ofPeptides IIH and V, Peptides IIH and V having the following amino acidsequences respectively: ##STR69## wherein X is --OH or --NH₂ or ananalogue of each of the above peptides having an amino acid sequencederived from a strain/isolate of HCV in a region corresponding to thepeptide and having specific immunoreactivity to antibodies to HCVrelative to the peptide that is substantially preserved.
 20. An ELISAtest kit according to claim 10 wherein the solid phase is coated with apeptide composition comprising a mixture of Peptides IIF, IIID and V,Peptide IIF, IIID and V having the following amino acid sequencesrespectively: ##STR70## wherein X is --OH or --NH2 or an analogue ofeach of the above peptides having an amino acid sequence derived from astrain/isolate of HCV in a region corresponding to the peptide andhaving specific immunoreactivity to antibodies to HCV relative to thepeptide that is substantially preserved.
 21. An ELISA test kit accordingto claim 10 wherein the solid phase is coated with a peptide compositioncomprising a mixture of Peptides IIH, V and VIIIE, Peptide IIH, V andVIIIE having the following amino acid sequences respectively: ##STR71##wherein X is --OH or --NH2 or an analogue of each of the above peptideshaving an amino acid sequence derived from a strain/isolate of HCV in aregion corresponding to the peptide and having specific immunoreactivityto antibodies to HCV relative to the peptide that is substantiallypreserved.
 22. An ELISA test kit according to claim 10 wherein the solidphase is coated with a peptide composition comprising a mixture ofPeptides VIIIE and IXD, Peptide VIIIE and IXD having the following aminoacid sequences respectively: ##STR72## wherein X is --OH or --NH₂ and ananalogue of each of the above peptides having an amino acid sequencederived from a strain/isolate of HCV in a region corresponding to thepeptide, and having specific immunoreactivity to antibodies to HCVrelative to the peptide that is substantially preserved; a segment ofeach of the above peptide or analogue thereof having specificimmunoreactivity to antibodies to HCV relative to the peptide of atleast 17.8% up to 101%.
 23. A peptide having the amino acid sequence:##STR73## wherein X is --OH or --NH₂.
 24. A peptide having the aminoacid sequence: ##STR74## wherein X is --OH or --NH₂.
 25. A peptidehaving the amino acid sequence: ##STR75## wherein X is --OH or --NH₂.26. A peptide having the amino acid sequence: ##STR76## wherein X is--OH or NH₂.
 27. A peptide having the amino acid sequence: ##STR77##wherein X is --OH or --NH₂.
 28. A peptide having the amino acidsequence: ##STR78## wherein X is --OH or NH₂.
 29. A peptide having theamino acid sequence: ##STR79## wherein X is --OH or --NH₂.
 30. A peptidehaving the amino acid sequence: ##STR80## wherein X is --OH or --NH₂.31. A peptide having the amino acid sequence: ##STR81## wherein X is--OH or --NH₂.
 32. A peptide having the amino acid sequence: ##STR82##wherein X is --OH or --NH₂.
 33. A peptide having the amino acidsequence: ##STR83## wherein X is --OH or NH₂.
 34. An ELISA test kit forthe detection of antibodies to HCV or NANBHV or the diagnosis of HCV orNANBHV infection comprising:(i) a solid phase coated with a peptidecomposition containing any one of the following peptides: ##STR84## ormixtures thereof; (ii) a negative control sample; and (iii) aninactivated HCV positive control sample; (iv) specimen diluent; (v)enzyme labelled antibodies to human IgG; and (vi) an enzyme substrate.35. An ELISA test kit for the detection of antibodies to HCV or NANBHVor the diagnosis of HCV or NANBHV infection comprising:(i) a solid phasecoated with a peptide composition comprising a mixture of ##STR85## (ii)a negative control sample; and (iii) an inactivated HCV positive controlsample; (iv) specimen diluent; (v) enzyme labelled antibodies to humanIgG; and (vi) an enzyme substrate.
 36. An ELISA test kit for thedetection of antibodies to HCV or NANBHV or the diagnosis of HCV orNANBHV infection comprising:(i) a solid phase coated with a peptidecomposition comprising a mixture of ##STR86## (ii) a negative controlsample; and (iii) an inactivated HCV positive control sample; (iv)specimen diluent; (v) enzyme labelled antibodies to human IgG; and (vi)an enzyme substrate.
 37. An ELISA test kit for the detection ofantibodies to HCV or NANBHV or the diagnosis of HCV or NANBHV infectioncomprising:(i) a solid phase coated with a peptide compositioncomprising a mixture of ##STR87## (ii) a negative control sample; and(iii) an inactivated HCV positive control sample; (iv) specimen diluent;(v) enzyme labelled antibodies to human IgG; and (vi) an enzymesubstrate.